Note: Descriptions are shown in the official language in which they were submitted.
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1333887
1 TITLE OF THE INVENTION
"IMMUNITY MEMORY CELL SUSPENSION AND METHOD OF PREPARING SAME"
FIELD OF THE INVENTION
The present invention relates to a method of preparing an
immunity memory cell suspension useful for the prevention
and/or treatment of an infectious disease or a malignant tumor
such as typically cancer. The present invention also relates
to an immunity memory cell suspension prepared by such a
method.
BACKGROUND OF THE INVENTION
A human being or other animal that has once experienced a
viral or bacterial disease and has reacted against a particu-
lar species of antigen exhibits a prominent defensive response
to the antigen when the individual is for a second time
subjected to stimulation with the same type of antigen. This
defensive reaction is caused as a result of the immunity
conferred to the individual against the particular disease and
is commonly called secondary immune response or anamnestic
response of an organism to the antigen. As well known in the
art, such an immune response of an organism to an antigen
results from excessive multiplication of the lymphocytes
involved in the reaction of the organism to the stimulation
with the antigen that has contributed to the outbreak of the
disease. The lymphocytes thus developed in response to the
stimulation by an antigen are generally called immunological
. . ~ . . ., ... ... . ... . . . ... . " , . . ~ ~ .. ; .
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1333887
1 memory cells.
Immunological memory cells produced in the body of an
organism are a species of lymphocytes inhabiting the lymph and
blood circulating through the vessels of the circulatory
system of the organism. The lymphocytes in the circulatory
system immigrate into the thymus gland of the organism where
they develop into T-lymphocytes or T-cells which confer an
immune ability to the organism. As well known in the art,
such immunologically competent T-cells, sometimes also called
thymus-dependent or thymus-derived cells, are distributed
extensively in almost all the peripheral tissues of the
circulatory system of an organism, as in the tissue of the
spleen, around the post-capillary venules in the paracortical
areas of the lymph nodes, and in the central artery of the
spleen white pulp of the organism.
As for the im.munity memory cells contained in the T-cells
thus developed from lymphocytes in the body of an organism,
evidence in the art seems to indicate that such cells are
recognized merely conceptually as ~immunological" memory cells
as above mentioned. There have indeed been no practical
attempts to isolate or extract such cells or at least any
fraction containing such cells from a tissue of an organism
and collecting the resultant immunity memory cells or cell-
cont~;n;ng fraction in a practically usable state or in vitro.
Neither a positive prospect for utilizing immllnnological
memory cells for the prevention and/or treatment of an
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1333887
1 infectious disease or a malignant tumor has ever been held out
or suggested on a realistic basis, nor any clinical or even
experimental approach toward such a practical use of immuno-
logical memory cells has been ambitiously undertaken.
The present invention contemplates realization of a
method of separating immunity memory cells directly from a
tissue of an organism at a significantly high efficiency.
Such a method according to the present invention depends for
its effect on various specific natures and attributes of
immunity memory cells which confer cell-mediated immunity to
an organism. The present invention further contemplates
provision of a method of cultivating immunity memory cells
through a plurality of generations to provide a continuous,
established cell line originating in the immunity memory cells
initially separated from a tissue of an organism.
SUMMARY OF THE INVENTION
It is accordingly an important object of the present
invention to provide a method of preparing a cell suspension
cont~;ning immunity memory cells separated from a tissue of an
organism that has been immunologically sensitized against a
bacterial or viral infectious disease or a malignant tumor
such as typically cancer.
It is another important object of the present invention
to provide a method of cultivating the immunity memory cells
separated from a tissue of an organism to establish a conti-
nuous line of immunity memory cells in vitro.
1333887
1 It is still another important object of the present
invention to provide a method of preparing an immunity memory
cell suspension from a tissue of an organism and thereafter
cultivating the immunity memory cells in the cell suspension
to provide an established line of immunity memory cells in
vitro.
It is still another important object of the present
invention to provide a cell line originating in the immunity
memory cells separated from a tissue of an immunity sensitized
individual in accordance with the present invention or in any
immllnity memory cells otherwise separated from a tissue of an
individual.
In accordance with one outstanding aspect of the present
invention, there is provided a method of preparing an immunity
memory cell suspension, comprising a) separating cells sub-
stantially consisting of T-cells from cells collected from an
immtlnity sensitized individual immunized against a particular
pathogen, b) adding to the separated cells an antibody reac-
tive to a histocompatibility antigen specific to a prescribed20 species of cells for producing antigen-antibody complexes by
. the antibody and those of the separated cells which have the
histocompatibility antigen, c) adding a blood serum to the
cells cont~i n; ng the antigen-antibody complexes for causing
the complement in the blood serum to bind to the antigen-
antibody complexes and thereby destroying the cells having the
histocompatibility antigen and allowing the remaining cells to
1333887
I persist, d) adding to the persisting cells an antibody reac-
tive to a histocompatibility antigen specific to immunity
memory cells, and e) separating from the resultant cells those
which have the histocompatibility antigen specific to immunity
memory cells.
In this method according to the first outstanding aspect
of the present invention, the cells which have the histocom-
patibility antigen specific to immunity memory cells may be
separated in the form of a first-stage immunity memory cell
suspension from the cells which have resulted from the addi-
tion of the antibody to the persisting cells. In this in-
stance, the method according to the first outstanding aspect
of the present invention further comprises f) preparing normal
cells syngeneic with the cells collected from the immunity
sensitized individual, g) exposing the normal cells to a
radioactive radiation for inhibiting the divisional ability of
the cells, h) preparing pathogenic cells of a pathogen of the
type identical with the particular pathogen, i) inactivating
the cells separated from the pathogen identical with the
particular pathogen, j) preparing a mixture of the first-stage
immunity memory cell suspension, the normal cells having their
divisional ability inhibited and the inactivated cells, and k)
incubating the mixture to allow the cells in the first-stage
immunity memory cell suspension to multiply and produce a
second-stage i~.unity memory cell suspension.
In accordance with another outstanding aspect of the
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1333887
1 resent invention, there is provided a method of preparing an
immunity memory cell suspension, comprising a) separating
cells substantially consisting of T-cells from cells collected
from an immunity sensitized individual immunized against a
particular pathogen, b) adding to the separated cells an
antibody reactive to a histocompatibility antigen specific to
a prescribed species of cells for producing antigen-antibody
complexes by the antibody and those of the separated cells
which have the histocompatibility antigen, c) adding a blood
serum to the cells containing the antigen-antibody complexes
for causing the complement in the blood serum to bind to the
antigen-antibody complexes and thereby destroying the cells
having the histocompatibility antigen and allowing the remain-
ing cells to persist, d) adding to the persisting cells
antibody molecules reactive to a histocompatibility antigen
specific to immunity memory cells, and e) preparing a first-
stage immunity memory cell suspension from the resultant
cells, the first-stage immunity memory cell suspension con-
taining cells having the histocompatibility antigen specific
to immunity memory cells, f) preparing normal cells syngeneic
with the cells collected from the immunity sensitized indi-
vidual, g) exposing the normal cells to a radioactive radia-
tion for inhibiting the divisional ability of the cells, h)
preparing pathogenic cells of a pathogen of the type identical
with the particular pathogen, i) inactivating the cells
separated from the pathogen identical with the particular
~ 1~33887
1 pathogen, j) preparing a mixture of the first-stage immunity
memory cell suspension, the normal cells having their divi-
sional ability inhibited and the inactivated cells, and k)
incubating the mixture to allow the cells in the first-stage
immunity memory cell suspension to multiply and produce a
second-stage immunity memory cell suspension
In a method according to the present invention as herein-
before described, the cells having the histocompatibility
antigens specific to immunity memory cells are separated
preferably by e/l) plating an anti-IgG antibody preparation
onto a surface to form a coating of the anti-IgG antibody
preparation on the surface, e/2) applying to the coating the
cells which have resulted from the addition of the antibody
molecules to the persisting cells so that the cells having the
histocompatibility antigens specific to immunity memory cells
adhere to the surface, and e/3) removing from the surface the
cells which have adhered thereto.
Furthermore, the cells substantially consisting of
T-cells as above mentioned are separated from the cells
collected from the immunity sensitized individual preferably
by a/l) subjecting the cells substantially consisting of
T-cells to a first stage of affinity column chromatography
using a solid adsorbent of glass wool, and a/2) thereafter
subjecting the resultant cells to a second stage of affinity
column chromatography using a solid adsorbent of nylon wool.
In accordance with still another outstanding aspect of
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1333887
1 the present invention, there is provided an immunity memory
cell suspension containing cells which are collected from an
~ nity sensitized individual immunized against a particular
pathogen and which substantially consist of T-cells, wherein
cells accounting about 98 per cent by number of the T-cells
contain at least lx103 ;mmtln;ty memory cells.
It may be noted that the term "pathogen" herein referred
to may be a cancerous or other malignant tumor or any patho-
genic microbe such as a bacterium or a virus that is known to
be causative of a disease or a toxic response in a human being
or other animal.
An immunity memory cell suspension prepared in a method
according to the present invention as hereinbefore described
can be separated, purified and cultured from any tissue of an
l~ organism that has been immunologically sensitized against an
infectious disease or a malignant tumor. This means that
immunological prevention and/or treatment of a disease can be
effected against any bacterial or viral infectious disease or
a malignant tumor insofar as an immunity sensitized org~ni cm
that has been cured of such a disease or tumor is available.
Typical of the diseases that can be coped with by such immN-
nological prevention and/or treatment are those caused by
pathogens including Cancer cells, Salmonella, Corynebacterum,
Pseudmbnas, Pasteurella, Streptococcus, ectromelia virus, and
2~ Sendai virus (mouse parainfluenza virus type 1).
It may also be noted that the immune mechanism evoked by
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1333887
1 an immunity memory cell suspension prepared in a method
according to the present invention basically differs from that
of the humoral immunity that depends on the antigen-antibody
reaction represented by vaccination. No such adverse effects
that are to inevitably result from vaccination could be
involved in the prevention and/or treatment of a disease by
the use of an immunity memory cell suspension proposed by the
present invention.
Where immunity memory cells provided by the present
n invention are cultivated from the cells obtained in the form
of the first-stage immunity memory cell suspension and further
subcultured successively through a plurality of generations
from the second-stage immunity memory cell suspension, a
continuous line of immunity memory cells can be established to
make it possible to manufacture such cells stably and econo-
mically on a quantity basis.
DETAILED DESCRIPTION OF THE lNv~NlION
For preparing an immunity memory cell suspension in
accordance with a first outstanding aspect of the present,
cells containing lymphocytes are first collected from an
individual (hereinafter referred to as immunity sensitized
individual) that is known to have been cured of and accord-
ingly immunized against any bacterial or viral infectious
disease or a cancerous or other malignant tumor. These
lymphocyte-containing cells may for example be extracted from
the peripheral blood collected from the heart or the vein of
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133~887
1 the immunity sensitized individual. Alternatively, the
lymphocyte-containing cells may be collected from a slice of
the immunity sensitized individual's spleen tissue.
T-cells are then separated from the lymphocyte-cont~ining
cells which have thus been collected from the peripheral blood
or the spleen tissue of the i~ nity sensitized individual
used. For this purpose, the lymphocyte-cont~ining cells are
processed by column affinity chromatography using a suitable
solid adsorbent allowing passage of T-cells selectively. Also
contained in the lymphocyte-containing cells herein in use are
B-cells which confer cell-mediated immunity to an organism.
These B-cells are in a major proportion caused to adhere to
the solid adsorbent and, as a consequence, a cell suspension
containing cells largely consisting of T-cells is fractionally
lS eluted from the column. Preferred as the solid adsorbent for
use in this column affinity chromatography process is nylon
wool packed in a column tube.
A solid adsorbent of nylon wool adsorbs not only B-cells
but also other unwanted cells such as typically macrophages
and leucocytes as well known in the art and, for this reason,
the affinity chromatography using a solid adsorbent of nylon
wool alone could not provide a satisfactorily high adsorption
efficiency for B-cells. In a method according to the present
invention, it is for this reason preferable that the affinity
chromatography process using the solid adsorbent of nylon wool
be performed subsequently to a column affinity chromatography
1333887
1 process using another solid adsorbent such as typically glass
wool. As well known in the art, both T-cells and B-cells
hardly adhere to a solid adsorbent of glass wool, which is
however capable of arresting macrophages and leucocytes with
significantly high degrees of affinity. Through use of the
two-stage affinity chromatography process using glass wool
prior to nylon wool, the B-cells in the initial lymphocyte-
containing cells can be removed at a satisfactorily high
efficiency from the cells which have been preliminarily
cleared of macrophages and leucocytes.
In substitution for such a two-stage affinity chromato-
graphy process may be used a T-cell separation process which
depends for its effect on the difference between the surface
antigens (histocompatibility antigens) specific to T-cells and
B-cells, respectively. Where a mouse is employed as the
immunity sensitized individual as previously noted, Thyl
surface antigen may be used to identify T-cells in such a
T-cell separation process.
The cells contained in the cell suspension resulting from
the two-stage column affinity chromatography process consist
largely of T-cells as above noted but still contain a small
quantity of unwanted species of cells such as typically
premature lymphocytes lacking in an immune ability. Accord-
ingly, the process to be performed subsequently is to remove
such unwanted premature cells by causing the cells to experi-
ence an antigen-antibody reaction using the cells in the cell
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1333887
1 suspension as the antigen.
For this purpose, an antibody which specifically reacts
with a particular histocompatibility antigen is added to the
cell suspension which has resulted from the two-stage column
affinity chromatography process. The antibody preparation to
be added to the cell suspension is selected to be capable of
reacting with the cells which have histocompatibility antigens
specific to the unwanted lymphocytes contained in the cell
suspension.
As a result of the antigen-antibody reaction thus caused
between the antibody preparation and the cells having such
histocompatibility antigens, antigen-antibody complexes are
formed by the antibody molecules united with the cells having
the histocompatibility antigens. To the antigen-antibody
complexes produced in this fashion is added a suitable blood
serum such as typically rabbit serum so that the complement in
the rabbit serum binds to the antigen-antibody complexes and
destroys the cells having the histocompatibility antigens
specific to the unwanted lymphocytes. The result is that only
the cells exhibiting a negative surface antigenic activity
against such histocompatibility antigens are allowed to
survive in the resultant cell suspension. It will be apparent
that these cells largely consist of immunity memory cells.
Where a mouse is used as the immunity sensitized individ-
ual as herein assumed to be the case, Ia (I-region-associatedJ
antigen may typically be used as the histocompatibility
.. ... .
... . . . .
1333887
1 antigen in the reaction to produce the antigen-antibody
complexes. In this instance, anti-Ia antigen monoclonal
antibody is added to the cell suspension to form the antigen-
antibody complexes by the Ia antigen molecules united with the
cells exhibiting a positive Ia antigenic activity. To these
antigen-antibody complexes is added a suitable blood serum so
that the complement in the serum binds to the
antigen-antibody complexes. The cells exhibiting the positive
Ia antigenic activity are thus destroyed and as a consequence
only the cells having a negative Ia antigenic activity are
allowed to persist in the resultant cell suspension. It may
be noted that the Ia antigen in mouse corresponds to the HLA-D
(or HLA-DR) histocompatibility antigen in man.
The cell suspension obtained at this stage consist of
cells which exhibit the negative surface antigenic activity
against the histocompatibility antigens specific to the
unwanted lymphocytes and which largely consist of immunity
memory cells. To such a cell suspension is further added an
antibody preparation typically containing rat antibody reac-
tive to the surface antigens specific to immunity memory
cells. Coatings of the rat antibody are formed on the surface
antigen molecules to produce antigen-antibody complexes by the
rat antibody formed on the molecules of the antigen specific
to the immunity memory cells.
To the immunity memory cells collected as a result of the
antigen-antibody and complement fixation reactions is then
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1333887
1 added an antibody, such as for example rat antibody, which is
reactive particularly to the surface antigens specific to
immunity memory cells. A coating of, for example, the rat
antibody is thus formed on the surface antigen of each immu-
nity memory cell.
Separately of the immunity memory cell suspension thusobtained is prepared an anti-rat IgG (gamma immunogloblin)
antibody preparation with use of a suitable anti-rat IgG serum
as a starting material. The anti-rat IgG antibody preparation
is applied to the inner surface of a glass vessel, into which
the cell suspension containing the immunity memory cells is
poured for reaction with the anti-rat IgG antibody plated in
the glass vessel. The anti-rat IgG antibody reacts with the
rat antibody wrapping the surface antigen on each immunity
memory cell and, accordingly, plays the role of an antigen in
the antigen-antibody reaction which is caused between the
anti-rat IgG antibody and the rat antibody. The result is
that the cells each having the rat antibody binding to the
surface antigen of the cell adhere to the inner surface of the
2~ glass vessel coated with the anti-rat IgG antibody suspension.
The cells which have failed to adhere to the inner
surface of the glass vessel are apparently lacking in the
ability to bind to the anti-rat IgG antibody on the surface of
the glass vessel and are thus considered to have no surface
antigens to form the antigen-antibody complexes that would
otherwise be for~ed by the rat antibody combined with the
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1333887
l surface histocompatibility antigens specific to ;mm1ln;ty
memory cells. This means that the cells not adherent to the
inner surface of the vessel substantially consists of cells
other than immunity memory cells and may be discarded as
useless from the glass vessel. In a method according to the
first outstanding aspect of the present invention, only the
cells which are deposited on the inner surface of the glass
vessel and which are accordingly considered to consist of
~ nity memory cells are therefore collected as useful cells.
The cell suspension contAining such useful cells is herein re-
ferred to as "first-stage" im~llnity memory cell suspension.
Known examples of the histocompatibility antigens specific to
immunity memory cells of a mouse include Thyl, Lytl, Lyt2,
Lyt3 and Lyt3 antigens.
1~ Tests conducted with the first-stage ir~nnity memory cell
suspension have revealed that the cells contained in the
first-stage ;mmllnity memory cell suspension account for
approximately 0.1 per cent of the cells in the cell suspension
resulting from the two-stage column affinity chromatography.
The results of the tests further indicate that the immunity
memory possessed by the cells in the first-stage immunity
memory cell suspension prepared in a method according to the
first outstanding aspect of the present invention is almost
perfectly transferred to a non-immunized individual when the
;mm~lnity memory cell suspension contains at least lx103
;mmllnity memory cells, as will be discussed in more detail.
, . . . . .. . .. . . . . .. . . .. . ... . .. . . . .
1333~7
Now, in accordance with a second outstanding aspect of
the present invention, immunity memory cells are cultivated
efficiently from the cells obtained in the form of the first-
stage immunity memory cell suspension prepared in accordance
with the first outstanding aspect of the present invention as
hereinbefore described. Immunity memory cells may thus be
cultured and subcultured successively through a plurality of
generations to establish a continuous line of cells which
originates in the "first-generation" immunity memory cells
separated from an immunity sensitized individual in accordance
with the first outstanding aspect of the present invention.
For cultivating cells or "second-generation" immunity
memory cells from the first-generation immunity ~emory cells,
normal diploid cells are first separated from a ~ouse synge-
neic with the mouse herein used as the immunity sensitizedindividual. These normal diploid cells are, after multipli-
cation, exposed to a suitable radioactive radiation such as,
for example, cobalt-60 gamma rays inhibiting the divisional
and multiplicative activity of the cells but allowing the
cells to live on. The normal diploid cells having their
divisional and multiplicative ability thus inhibited are
herein referred to as mitosis-inhibited normal diploid cells.
On the other hand, pathogenic cells of the type that has
caused the disease contracted by the immunity sensitized
individual herein in use are inactivated to produce an inacti-
vated pathogenic antigen suspension as herein so referred to.
.
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1 These pathogenic cells are used as the antigen of the type
memorized by the immunity memory cells now provided in the
form of the first-stage immunity memory cell suspension. For
this purpose, such pathogenic cells are collected from an
5 individual presently contracted by a disease caused by the
pathogen under consideration is suspended in a culture medium
and the resultant cell suspension is subjected to electro-
magnetic or radioactive radiation with, for example, ultra-
violet or cobalt-60 gamma rays.
The mitosis-inhibited normal diploid cells are then added
to the first-stage immunity memory cell suspension which has
been prepared as hereinbefore described. To the mixture of
these is further added the inactivated pathogenic antigen
suspension. The resultant mixture is incubated to promote
multiplication of the im~unity memory cells in the suspension
to thereby yield a n second-stage" immunity memory cell suspen-
sion in a method according to the second outstanding aspect of
the present invention. It may be herein noted that this
second-stage immunity memory cell suspension is the product of
the primary culture of the first-generation immunity memory
cells separated from the immunity sensitized individual that
is herein assumed to be in use. It may be further noted that
immunity memory cells of a subsequent generation can be easily
subcultivated when the second-stage immunity memory cell
suspension prepared as above described is processed in a
manner similar to that used for the primary culture of the
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133388~
l cells from the first-stage immunity memory cell suspension.
Thus, immunity memory cells of the third generation can be
obtained with the resultant mixture incubated under conditions
similar to those used for the cultivation of the second-
generation immunity memory cells from the first-generation
immunity memory cells. Immunity memory cells according to the
present invention can be in these manners easily cultivated
from one generation to another to establish a strain of
immunity memory cells at a satisfactorily high efficiency when
a procedure similar to that used for the cultivation of the
immunity memory cells of the second generation from the those
of the first generation is followed repeatedly.
According to the results of the experiments conducted by
the inventor, the immunity memory cells according to the
1~ present invention multiply at a rate which, in terms of the
number of cells, increases with a factor of approximately
three for five days of incubation during cultivation of the
cells (the second-generation immunity memory cells) starting
with the cells obtained by the primary culture. Such a high
rate of multiplication of cells will provide an assurance of
creating an established line of immunity memory cells and
manufacturing such cells stably and economically on a quantity
basis.
It may be further noted that the immunity memory cells of
2~ the seccnd generation cultivated from those of the first
generation appear generally globular in shape and are
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1333887
1 approximately three times larger than the first-generation
immunity memory cells when observed microscopically. It may
be added that the second-generation immunity memory cells have
nuclei almost all of which are larger in size than those of
the first-generation immunity memory cells and capsular
antigen molecules which are largely identical with those of
the first-generation immunity memory cells.
Tests were further conducted with the second-stage
immunity memory cell suspension, the results of the tests
indicating that the immunity memory possessed by the cells in
the second-stage immunity memory cell suspension prepared in a
method according to the second outstanding aspect of the
present invention is also almost perfectly transferred to a
non-immunized individual when the immunity memory cell suspen-
t5 sion contains at least lx103 immunity memory cells, as will bediscussed in more detail.
A method of preparing an immunity memory cell suspension
in accordance with the present invention as hereinbefore
described may be put into practice in any of various manners
which fall within the purview of the present invention.
Hereinafter described is a preferred example of such various
manners of carrying out a method according to the first and
second outstanding aspects of the present invention.
EXAMPLE
(l) Separating Immunity Memory Cells from Organism
A mouse recovered from and therefore immunologically
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1333887
1 sensitized against salmonellosis was used as the immunity
sensitized individual in this Example. It will be however
apparent that, if desired, a mouse immunized against any other
bacterial infectious disease or cured of any viral infectious
disease or a cancerous or other malignant tumor may be used in
substitution for the immunity sensitized individual herein
selected for use.
Preparing Initial Cell Suspension
(1-1) Peripheral blood was collected from the heart of
the mouse for use as the starting material in this Example.
The blood collected was diluted with a five-fold volume of
Hanks' solution to dissociate the cell aggregates into a
suspension of single cells, followed by centrifugation at 2000
rpm for 15 minutes at 4C. A buffy coat formed by the super-
natant in the resultant preparation was removed from theremainder of the preparation by gently sucking in the super-
natant.
(1-2) The buffy coat fraction, containing leucocytes in a
major proportion, was further diluted with a five-fold volume
of Hanks' solution. The resultant cell suspension was centri-
fuged at 2000 rpm for 15 minutes at 4C, followed by removal
of the supernatant. The cell suspension thus obtained was
further centrifuged in two steps each under conditions similar
to those used for the immediately earlier step of centrifuga-
tion. The supernatant formed on the surface of the cellsuspension produced during each of these two steps of
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1333887
1 centrifugation was removed at the end of each of the
centrifugation steps. To the cells which had been washed
through the total of three steps of centrifugation each
followed by removal of the supernatant was added a Hanks'
solution in a proportion selected to yield a cell density of
4x107 cells/ml and containing 5% by volume of fetal cow serum
(FCS). The cell suspension obtained at this stage of
processing will be hereinafter referred to as "initial" cell
suspension.
n (1-3) In substitution for the peripheral blood herein
used as the starting material may be used the spleen tissue of
an immunity sensitized individual if desired. Where the
spleen tissue of a mouse is used as the starting material, a
slice of spleen tissue is excised from the mouse's spleen and
is forced through a steel strainer having perforations con-
forming to, for example, Tyler standard screen scale No. 100.
The cells passed through the perforations in such a steel
strainer are dispersed into an appropriate volume of Hanks'
solution and may be thereafter processed similarly to the cell
suspension prepared from the peripheral blood of the mouse
herein assumed to be used. Thus, the cell suspension con-
taining the spleen-derived lymphocyte-containing cells may
also be centrifuged at 2000 rpm for 15 minutes at 4C, fol-
lowed by two further steps of centrifugation each under
conditions similar to those used for the first step of centri-
fugation of the cell suspension prepared from the peripheral
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1 blood of the mouse as previously described.
Separating T-Cells
(1-4) The initial cell suspension prepared from the
peripheral blood of the mouse as hereinbefore described was
applied to a glass wool column having 10 grams of glass wool
packed in a vertical glass tube (measuring 20mm in inside
diameter and lOOmm in length), followed by addition of a
Hanks' solution containing 5% by volume of fetal cow serum.
The cell suspension was then heated in the glass wool column
at 37C for 45 minutes for incubation of the cells in the
suspension. After the incubation, the cell suspension was
allowed to slowly drip from the column tube with a similarly
proportioned Hanks' solution being continuously added to the
column which was maintained at 37C. The cell suspension thus
fractionally eluted from the glass wool column was collected
in a centrifuge tube.
(l-S) As previously noted, T-cells and B-cells tend to
adhere to fibers of glass wool less firmly than other types of
cells do and, for this reason, cells other than T-cells and,
generally, B-cells contained in a cell suspension added to a
glass wool column are selectively retained to the fibers of
the glass wool. This means that the cells contained in the
cell suspension resulting from the glass wool column affinity
chromatography used in this Example largely consisted of
T-cells and B-cells. Typical of the cells thus isolated from
the T-cells and B-cells and left in the column are macrophages
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1333887
and leucocytes as previously noted.
(1-6) Tests were conducted with the cell suspension
collected by the glass wool column affinity chromatography
process as hereinbefore described. The results of the tests
indicated that the T-cells and B-cells separated through the
glass wool column affinity chromatography accounted for
approximately 30 per cent of the total quantity of the cells
which had been contained in the initial cell suspension
applied to the glass wool column.
1 (1-7) Subsequently to the glass wool col D affinity
chromatography, the cell suspension collected in the centri-
fuge tube was subjected to centrifugation at 2000 rpm for 15
minutes at 4C, followed by removal of the supernatant from
the resultant suspension. To the cell suspension thus ob-
tained was added a Hanks' solution in a proportion selected to
yield a cell density of 4x107 cells/ml and containing 5% by
volume of fetal cow serum. The resultant preparation was
applied to a nylon wool column having 10 grams of nylon wool
(manufactured by Fenwal Laboratories) packed in a vertical
glass tube (measuring 20mm in inside diameter and lOOmm in
length), followed by addition of a Hanks' solution containing
5% by volume of fetal cow serum. The cell suspension was then
heated in the nylon wool column at 37C for 45 minutes for
incubation of the cells in the suspension. After the incuba-
tion, the cell suspension was allowed to drip from the column
tube with a similarly proportioned Hanks' solution being
.. . . .. . ..
- 24 -
1333887
1 continuously added to the column which was maintained at 37C.
The cell suspension thus fractionally eluted from the nylon
wool column was also collected in a centrifuge tube.
(1-8) Tests were conducted with the cell suspension
collected as a result of this nylon wool column affinity
chromatography, the results of which indicated that the cells
separated through the nylon wool column affinity chromato-
graphy accounted for approximately 10 per cent of the total
quantity of cells which had been contained in the initial cell
suspension applied to the glass wool column.
(1-9) The cells contained in the cell suspension result-
ing from the two-stage column affinity chromatography process
contain T-cells in a major proportion which, according to the
results of the tests conducted, amounted to approximately 95
per cent of the total cell population in the cell suspension.
Most of those cells other than the T-cells that had been
contained in the initial cell suspension prior to the two
stages of column affinity chromatography are considered to
have been adsorbed partially to the fibers forming the glass
wool and partially to the fibers forming the nylon wool. As
noted previously, however, the cell suspension obtained
through the two-stage column affinity chromatography process
still contains lymphocytes and other cells unwanted in a
method according to the present invention.
Purifying T-Cells
(1-10) For the purpose of removing these unwanted
1333887
l species of cells, the cell suspension collected in the centri-
fuge tube as a results of the nylon wool column affinity
chromatography was subjected to centrifugation at 2000 rpm for
15 minutes at 4C, followed by removal of the supernatant
therefrom. The resultant cell suspension was added to a
culture medium in a proportion selected to yield a cell
density of 8x107 cells/ml. The culture medium herein used
consisted of a mixture of Medium 199 containing 2~ by volume
of fetal cow serum. A culture medium having such a composi-
tion will be hereinafter referred to as medium "A".
(l-11) To one part by volume of the cell suspension thus
obtained were then added two parts by volume of an anti-Ia
(anti-I-region-associated) antigen monoclonal antibody prepa-
ration (manufactured by Cedarlane Laboratories Ltd.) diluted
with a thirty-fold volume of medium "A" and one part by volume
of rabbit serum diluted with a two-fold volume of medium "An.
The mixture of these three component preparations was heated
for reaction at 37C for 30 minutes.
(1-12) As a result of the reaction thus caused in the
mixture, antigen-antibody complexes were formed by the anti-Ia
monoclonal antibody united with the cells having a positive Ia
antigenic activity. To the resultant mixture was added rabbit
serum so that the complement in the rabbit serum bound to the
antigen-antibody complexes. The cells having the positive Ia
antigenic activity were thus destroyed by the rabbit serum
and, accordingly, only the cells having a negative Ia
.. , , . . . , ~, .. ..
- 26 -
-
1333887
1 antigenic activity were allowed to survive. The cell
suspension, now containing a concentrated population of
immunity memory cells, was centrifuged at 2000 rpm for 15
minutes at 4C to collect the immunity memory cells.
(1-13) Centrifugation was further performed for the
resultant cell suspension in three successive steps each at
2000 rpm for 15 minutes at 4C in medium "A". The cell
suspension obtained at the end of the three steps of centri-
fugation was diluted with medium "A" in a proportion selected
to yield a cell density of 8x107 cells/ml.
(1-14) Besides this cell suspension, an anti-immunity
memory cell rat antibody suspension was prepared in a manner
to be described (chapter (4)) and was diluted with a five-fold
volume of medium "An. The rat antibody suspension and the
cell suspension each prepared as described above were mixed
together in substantially equal volumes and the resultant
mixture was allowed to react at 4C for 60 minutes. As a
result of this reaction, the rat antibody bound to the antigen
molecules specific to immunity memory cells. Antigen-antibody
complexes were produced by the coatings of the rat antibody
formed on the antigen molecules specific to immunity memory
cells. The cell suspension containing the immunity memory
cells carrying such antigen-antibody complexes was centrifuged
at 2000 rpm for 15 minutes at 4C in medium "A". Centrifuga-
tion was further performed on the resultant cell suspension inthree successive steps each under conditions similar to those
- 1333887
1 used for the preceding step of centrifugation. To the cells
obtained through these steps of centrifugation was added
medium "A" in a proportion selected to yield a cell density of
8X106 cells/ml.
(1-15) In addition to this immunity memory cell suspen-
sion was prepared an anti-rat IgG goat antibody preparation.
For this purpose, 10 ml of anti-rat IgG goat serum diluted
with a thirty-five fold volume of medium "A" was prepared in a
sterile plastic petri dish (manufactured by Becton Dickinson
Co., Ltd.) measuring lOOmm in inside diameter and was allowed
to stand overnight at 4C. An excess of antiserum was then
washed away from the petri dish so that an anti-rat IgG goat
antibody was allowed to remain uniformly in the dish in the
form of a thin liquid coating deposited on the inner surface
15 of the dish.
(1-16) The immunity memory cell suspension prepared as
described above was poured into the petri dish having the
liquid coating of the anti-rat IgG goat antibody deposited
therein. The petri dish was then maintained horizontally and
20 was allowed to stand undisturbed for 60 minutes. Thereupon,
the cells which had failed to adhere to the coating on the
inner surface of the dish were removed and discarded from the
dish by trembling the dish lightly. As has been noted, the
cells that had failed to bind to the coating of the anti-rat
25 IgG goat antibody have no antigen-antibody complexes which
would have otherwise been formed by the rat antibody combined
., . , .. . . . , , . . . .. . , j . . . ., ~ .. ~ .
- 28 -
_.
1333887
1 with the antigens specific to ;mmlln;ty memory cells. Most of
the cells floating on the coating of the anti-rat IgG goat
antibody on the surface of the dish are thus considered to
consist of non-;mmlln;ty memory cells.
(1-17) The inner surface of the dish was then rinsed in
three steps each with a mixture of Medium RPMI 1640 containing
10% by volume of fetal cow serum, followed by spraying of the
same medium over the inner surface of the petri dish. The
cells which adhered to the inner surface of the petri dish
were in this manner stripped from the surface of the dish and
were collected as the first-stage immunity memory cell suspen-
sion in a method according to the present invention.
(1-18) The cells collected at this stage of the process
accounted for approximately 0.1 per cent of the cells that had
1~ been contained in the cell suspension resulting from the two
successive stages of column affinity chromatography. Tests
were conducted with this first-stage immunity memory cell
suspension, the results of which showed that the cells ac-
counting for about 98 per cent by number of the cells con-
2~ tained in the first-stage immllnity memory cell suspension
(1) belong to a family of small lymphocytes;
(2) exhibit a positive antigenic activity in respect of
the antigen molecules specific to immunity memory cells; and
(3) exhibit a positive antigenic activity in respect of
25 the Thyl, Lytl, Lyt2 and Lyt3 antigens and a negative
antigenic activity in respect of the Ia antigen.
.
_ - 29 -
1333887
l (1-19) Tests were further conducted wherein the immunity
memory cells in the form of the first-stage immunity memory
cell suspension were introduced into a non-immunized individ-
ual syngeneic with the mouse herein used as the immunity
sensitized individual. The results of the tests showed that
the immunity memory possessed by the immunity memory cells was
transferred to the recipient perfectly, viz., with a probabil-
ity of 100 per cent when the immunity memory cell suspension
contains at least lx103 immunity memory cells, as will be
described in more detail. Tests were further conducted with
the first-stage immunity memory cell suspension, wherein
individuals into which the immunity memory cells had been
introduced were irradiated with gamma rays from cobalt-60.
The results of these tests indicated that the immunity memory
which each of the individuals had acquired remained unimpaired
when the individual was exposed to cobalt-60 gamma rays of 600
roentgens or less but was destroyed when the individual was
subjected to irradiation with cobalt-60 gamma rays of more
than 1100 roentgens. This evidences that the immunity memory
cells contained in the first-stage memory cell suspension
prepared in this Example belong to a family of cells which are
significantly resistant to radioactive radiations.
(2) Cultivating Cells for Establishing Cell Line
Cells were then cultivated from the immunity memory cells
obtained in the form of the first-stage immunity memory cell
suspension prepared as hereinbefore described. It may be
- - 30 -
- 13338~7
1 noted that the steps of cultivating cells as will be herein-
after described are useful not only for the establishment in
vitro of a cell line originating in the immunity memory cells
contained in the first-stage immunity memory cell suspension
prepared in accordance with the first outstanding aspect of
the present invention but also for the culture and subculture
of immunity memory cells in general which may be otherwise
separated or extracted from an immunity sensitized individual.
Cultivating Syngeneic Normal Cells
(2-1) Normal diploid cells were separated in the form of
a cell suspension from a mouse syngeneic with the mouse used
as the immunity sensitized individual in this Example and were
grown in a sterile petri dish. Approximately when it was
observed that a single layer of cells was formed in the petri
1~ dish, the cells were exposed to cobalt-60 gamma rays of 2000
roentgens to inhibit the divisional and multiplicative ability
of the cells.
Preparing Inactivated Pathogenic Antigen
(2-2) On the other hand, pathogenic cells of the type
that caused the disease (salmonellosis) contracted by the
immunity sensitized individual herein used was inactivated to
produce an inactivated pathogenic antigen suspension. For
this purpose, bacterial cells of Salmonella were added to a
culture medium in a proportion selected to yield a cell
density of lx107 cells/ml. The culture medium used for this
prccess consisted of a mixture of Medium RPMI 1640 containing
1333887
1 10% by volume of fetal cow serum, 2 grams/liter of sodium
hydrogencarbonate (NaHCO3), 0.11 gram/liter of sodium pyruvate
(NaOOCCOCH3), and 0.1% by volume of yeast oleate. A culture
medium having this composition will be hereinafter referred to
as medium "Bn.
(2-3) The suspension containing the bacterial cells of
Salmonella dispersed in the medium "B" was irradiated with
ultraviolet rays of an intensity of 3.6x103 erg/cm2.sec for
five minutes to inactivate the bacteria. The use of medium
"B" in this inactivation process is merely by way of example
and, as such, any other medium having a desired composition
may be used in substitution for medium "Bn.
(2-4) Where an ;mmunity sensitized individual that has
contracted a viral infectious disease is used for the separa-
tion of immunity memory cells therefrom, virus crystals may bedispersed in medium "B" in a proportion selected to yield a
density of lx10 crystals/ml.
(2-5) On the other hand, if it is desired to use malig-
nant tumor cells such as cancer cells, a slice of a tissue
involving tumor is excised from the body of an individual and
is cut into fragments with use of a knife or scissors. The
fragments of the tumor cell tissue may then be immersed in a
phosphate buffered physiological salt solution containing
0.02% by volume of ethylenediaminetetraacetic acid (EDTA) and
agitated for 60 minutes at 4C. The resultant pieces of
tissue may be pressed against a steel strainer having
- 32 -
- 1333887
l perforations conforming to, for example, Tyler standard screen
scale No. 100. The cell suspension containing the cells
forced through the perforations in the strainer is centrifuged
at 700 rpm to 800 rpm for five minutes at 4C. The tumor
cells collected in this manner is dispersed into medium "B" in
a proportion selected to yield a cell density of 2x103
cells/ml and the resultant cell suspension is exposed to
cobalt-60 gamma rays of 2000 roentgens to produce an inacti-
vated pathogenic antigen suspension equivalent to that used in
this Example. It will be apparent that, where an inactivated
pathogenic antigen suspension is to be prepared from a tumor
cell tissue, any desired culture medium other than medium "B~
may be used for the processing of the cells extracted from the
tissue.
Cultivating Tmmlln;ty Memory Cells
(2-6) To the first-stage immunity memory cell suspension
which had been prepared as hereinbefore described was then
added medium "B" in a proportion selected to yield a cell
density of 8x105 cells/ml. The resultant cell suspension was
gently poured into the petri dish cont~;n;ng the mitosis-
inhibited cells separated from the syngeneic mouse. The
volume of the cell suspension thus added to the mitosis-
inhibited cells may be selected depending on the size of the
petri idish used and was, in this Example, selected at S
2~ milliliters in consideration of the size of the petri dish
which measured lOOmm in inside diameter. If a petri dish
, . . . . .. . . . . . . . . .. .
- - 33 -
- 13338g7
1 having another inside diameter of, for example, 60mm is to be
used, it is advisable to use 2.5 milliliter of the first-stage
immunity memory cell suspension in the dish.
(2-7) To the mixture of the immunity memory cell suspen-
sion and the mitosis-inhibited cells plated in the petri dish
was added an equal volume of the inactivated pathogenic
antigen suspension which had been prepared as described above.
The resultant mixture was incubated at 37C in an atmosphere
containing 5% of carbon dioxide to promote multiplication of
the immunity memory cells in the mixture. The preparation
resulting from this centrifugation is the second-stage immu-
nity memory cell suspension prepared in a method according to
the second outstanding aspect of the present invention.
(2-8) When the second-stage immunity memory cell suspen-
sion prepared as hereinbefore described is processed in amanner similar to that used for the primary culture of the
cells from the first-stage immunity memory cell suspension,
immunity memory cells of a subsequent generation can be easily
subcultivated, as previously noted. For this purpose, the
second-stage immunity memory cell suspension may be diluted
with medium "B" in a proportion selected to yield a cell
density of 8x105 cells/ml. The resultant cell suspension is
mixed with the mitosis-inhibited cells of the syngeneic
individual and the inactivated pathogenic antigen suspension
prepared as previously described. Immunity memory cells of
the subsequent, viz., third generation can thus be obtained
.. . . . . ..
- 34 -
- 133388~
l when the resultant mixture is incubated and centrifuged under
the specified conditions. As has been noted, the experiments
conducted by the inventor have revealed that, during cultiva-
tion of the ; mm~n; ty memory cells starting with the cells
5 obtained by the primary culture, the immunity memory cells
multiply at a rate which, in terms of the number of cells,
increases with a factor of approximately three for five days
of incubation.
(3) Preventive Effect of Tm~t~n;ty Memory Cells
Tests were conducted with ;mmll~; ty memory cell suspen-
sions each prepared in a method according to the present
invention for assaying the disease preventive effects of the
cells against various pathogens. The cell suspensions used
for the assay include those represented by the first-stage
5 imm~ln;ty memory cell suspension cont~;n;ng ;mm~ln;ty memory
cells separated from an ;mmlln;ty sensitized individual and
those represented by the second-stage i-m-~unity memory cell
suspension containing ;mmlln;ty memory cells grown from the
cells in the first-stage immunity memory cell suspension. The
2~-~ cell suspensions used for the tests contained different
quantities of ; mmlln; ty memory cells ranging in nu-mber from
lx102 to lx104. These cell suspensions were introduced into
non-;mmllni7ed mice as recipient individuals and, thereafter,
the re`cipient individuals were attacked by different pathogens
to evaluate the disease preventive effects in percentage. The
pathogens which were used in the tests include cancer cells,
- 35 -
1333887
1 Salmonella, Corynebacterum, Pseudmonas, Pasteurella,
Streptococcus, ectromelia virus and Sendai virus. These
bacterial, viral and cancer cell pathogens were introduced
into the individuals in quantities selected to provide the
incidence rate of 100% for each of the pathogens. The results
of the tests are demonstrated in the following table.
Pathogens No. of CellsPreventive Effect
Cancer cells 1 x 104 100%
1 x 103 100%
6 x 102 70%
3 x 102 42%
1 x 102 34%
Salmonella 1 x 10 100%
1 x 103 100%
6 x 10 68%
3 x 1o2 34%
1 x 102 20%
Corynebacterum 1 x 10 100%
1 x 103 100%
6 x 10 48%
3 x 102 26%
1 x 102 14%
Pseudmonas 1 x 104 100%
1 x 103 100%
6 x 102 41%
3 x 102 23%
1 X 102 11%
_ - 36 -
1333887
1 Pasteurella 1 x 104 100%
1 x 103 100%
6 x 102 78%
3 x 1o2 53%
1 x 102 35%
Streptococcus 1 x 10 100%
1 x 103 100%
6 x 102 44%
3 x 102 31%
1 x 102 17%
ectromelia virus 1 x 104 100%
1 x 103 100%
6 x 102 51%
3 x 102 27%
1 x 102 13%
Sendai virus 1 x 104 100%
1 x 103 100%
6 x 102 88%
3 x 102 63%
1 x 102 29%
As will be understood from these results of the tests
conducted, the immunity memory possessed by the cells in a
immunity memory cell suspension prepared in a method according
to the present invention is successfully transferred to a
non-immunized individual with the probability of 100% when the
immunity memory cell suspension contains lx103 immunity memory
cells or more. The results of the tests further indicate that
.. .. .
_ ~ 37 ~ 1333 88~
1 considerably high degrees of disease preventive effect can be
achieved when an immunity memory cell suspension containing
less than lx103 immunity memory cells is introduced into an
individual. For example, when an immunity memory cell suspen-
sion containing less than 6X102 immunity memory cells isintroduced into a non-immunized individual, the imml7n;ty
memory is transferred to the recipient individual with the
probability of about 50% and, if an immunity memory cell
suspension containing less than 3X102 immunity memory cells is
used, the immunity memory is transferred to the recipient
individual with the probability of about 30%, as will be seen
from the results of the tests indicated in the table.
(4) Preparing Anti-Memory Cell Rat Antibody
Description will be hereinafter made regarding the
process of preparing the anti-immunity memory cell rat anti-
body suspension used in the steps hereinbefore described in
paragraph 1-14.
For this purpose, the immunity memory cell suspension
prepared as described in paragraph 1-13 was diluted with
phosphate buffered physiological salt solution and the resul-
tant preparation was centrifuged under appropriate conditions.
The cell suspension obtained by three steps of centrifugation
was, after washing, further diluted with similar salt solution
in a proportion selected to yield a cell density of lx108
cells/ml. A 0.25 ml fraction of the resultant immunity memory
cell suspension was injected into the femoral subcutaneous
.. . . . , . . , . , , . ~ . . .. . . . .. ~., . ~ . ..
_ - 38 -
1333887
1 tissue of each of a plurality of female rats (of Wister
species). In ten days and twenty days, respectively, after
the cell suspension was thus administered to each rat, similar
fractions of the cell suspension were injected into the
femoral subcutaneous tissue of the rat.
The ;mm-lnity memory cell suspension diluted with phos-
phate buffered physiological salt solution and centrifuged in
three successive steps as described above was also diluted
with phosphate buffered physiological salt solution this time
in a proportion selected to yield a cell density of lx109
cells/ml. A 0.25 ml fraction of the resultant immunity memory
cell suspension was injected into the abdominal cavity of each
of the female rats in forty days after the cell suspension had
been administered to the rat for the first time. A total
volume of blood was then collected from each of the rats in
fifty to sixty days after the cell suspension had been for the
first time administered to the rat. Five to six milliliters
of blood sera were obtained from each of the rats.
On the other hand, a slice of spleen tissue was excised
from a newborn mouse syngeneic with the mouse from which the
immunity memory cells had been collected. The slice of the
spleen tissue was forced through a steel strainer and the
spleen cells passed through the steel strainer were dispersed
into phosphate buffered physiological salt solution. The cell
suspension thus prepared was centrifuged in three successive
steps and, thereafter, an approximately 1.5 part by volume of
. .
_ - 39 -
- 1333887
1 the rat serum prepared as described above was added to the
resultant population of cells, followed by thorough agitation.
The preparation thus obtained was then allowed to stay in iced
water for 60 minutes with agitation at time intervals of 15
minutes and was thereafter centrifuged under appropriate
conditions.
From the resultant preparation was then collected the
supernatant, which was diluted with phosphate buffered physio-
logical salt solution, followed by steps similar to those10 taken for the cell suspension initially prepared from the
spleen tissue. Supernatant was further collected from the
preparation thus obtained finally and was used as the anti-
immunity memory cell rat antibody preparation in the steps
hereinbefore described in paragraph 1-14.
From the foregoing description it will have been under-
stood that an immunity memory cell suspension to be prepared
in a method according to the present invention can be sepa-
rated, purified and cultured from any tissue of an organism
that has been immunologically sensitized against an infectious
disease or a malignant tumor. Immunological prevention and/or
treatment of any bacterial or viral infectious disease or a
malignant tumor or tumor can be effected if an ~ n;ty
sensitized organism that has cured of the disease or tumor is
practically available.
It is also important that the im~une mechanism evoked by
an immunity memory cell suspension prepared in a method
. , ~ . . , . ,, ~ ~ . . . . ....
- 40 -
1333887
1 according to the present invention basically differs from that
of the humoral immunity that depends on the antigen-antibody
reaction represented by vaccination. No such adverse effects
that are to inevitably result from vaccination could be
involved in the prevention and/or treatment of a disease by
the use of an immunity memory cell suspension proposed by the
present invention.
It may be further added that where immunity memory cells
provided by the present invention are cultivated from the
cells obtained in the form of the first-stage immunity memory
cell suspension and further subcultured successively through
generations from the second-stage immunity memory cell suspen-
sion, a continuous line of immunity memory cells can be
established to make it possible to manufacture such cells
stably and economically on a quantity basis.
. .... . ... , ~ .. . ... . .... . .
