Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
L9~1~
CANCER CELL-CO~IBATTING LY~IPHOCYTES~ PROCESS
FOR TI~E PRODUCTION THEREOF, AND ANTICANCER
AGENTS CONTAINING SAID LYMPHOCYTES
FIELD OF rHE INVENTION
The present invention relates to cancer cell-
combatting lymphocytes (hereinafter referred to as "killer
cells") and a process for the production thereof More
palticularly it is concerned wi-th killer cells which act
specifically on cancer cells having a glycorelated antigen
deri~ed from the cancer cells (this antigen is hereinafter
lefeTred to as "GRA") and destroy the cancer cells, and a
process for the production of said killer cells. Additionally
the p~esent invention relates to a novel anticancer agent,
more specifically containing the killer cell or GRA as an
active ingredient.
BACKGROUND OF THE INVE~TION
1~ It i5 known that immune response effector cells,
particularly T lymphocytes playing a main role in cell-
medl~ted immune response, cause rejeotion of grafts due to
~oleign cell antigens, but exhibit no appreciable or very
limited immuno-inhibition against cancer cells. Thus, the
~O cance-r cells are not destroyed and multiply in vi~o, finally
putting the cancer-bearing host to death.
. ~
However, the mechanism responsiole to the recogni-
tion of self from not self is not completely clear and various
investigations have been made to have insight into the nature
of material basis involved in swch system. For example, cell
surface markers on mouse leukemia cells or embryonal carcinoma
cells using lectins such as Dolichos biflorusagglutinin (DBA)
and peanut agglutinin (PNA) as described in Biochem. Biophys.
~es. Comm. 89 (~) 448-455 (1979), Ibid. 96 (4) 1547-1553 (1980),
J. Biochem. _ 473-481 (1981) and Cell 18 183-191 September
1979.
However, as far as is known, there have been no
appreciable attempts to provide new lymphocytes that can combat
cancer cells specifically and also provide anticancer agents
utilizing immune response of lymphocytes.
SUMMARY OF THE INVENTION
As a result of extensive studies on the immune
response of the host to cancer cells and its application to
the treatment of cancer, it has been found that, in a cancer
cell specific antigen which is not found in differentiated
normal cells, there is GRA which acts as an immunogen for
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the host and have very high immunogencity that cause an
immune response specific to the cancer cells. Furthermore,
it has been found that when GRA is used to sensitize lympho-
cytes, ther~ can be obtained killer cells which act speci-
fically on cancer cells containing GRA and if the killercells are administered to the host, they recognize GRA and
act on the cancer cells containing GRA~ destroying them, and
thus ~hat they exhibit an excellent effect in ~he treatment
and prevention of cancer.
Therefore, the present invention provides killer
cells in one embodiment thereof.
In another embodiment, the present invention pro-
vides a process for the production of killer cells.
In still another embodiment~ the present invention
provides an anticancer agent containing a killer cell or GRA
as an active ingredient.
BRIEF DESCRIPTION OF THE DRAWINGS
_
Fig. l is a microphotograph of Daudi cancer cells;
Fig. 2 is a microphotograph lllustrating the forma-
tion of plaques by GRA-l-K-T of the cancer cell of Fig. l;
Fig. 3 is a microphotograph of KAIO-III cancer
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, . .... ..... .. ~.t.. ,
~z~g~
cells;
Fig. 4 is a microphotograph illustrating the forma-
tion of plaques by GRA-l-K-T of the cancer cell of Fig. 3;
Fig. 5 is a microphotograph of BT-I cancer cells;
Fig. 6 is a microphotograph showing -thé formation
of plaques by GRA-l-K-T of the cancer cell of Fig. 5;
Fig. 7 is a microphotograph of MKN-45 cancer cells;
Fig. 8 is a microphotograph showing the formation
of plaques by GRA-l-K-T of the cancer cell of Fig. 7;
Fig. 9 is a microphotograph of MOLT cancer cells;
Fig. 10 is a microphotograph showing the formation
of plaques by GRA-l-K-T of the cancer cell of Fig. 9;
Fig. 11 is a microphotograph of BT-l treated with
a mixture of unsensitized human peripheral blood lymphocytes;
Figs. 12 and 13 are each a microphotograph of the
cancer cell tissue of cancer-bearing mouce which is adminis-
tered with GRA-M-l-K;
Fig. 14 is a microphotograph showing the state of
cancer in a cancer-bearing mouse group which is administered
with GRA-M-l;
-Fig. lS is a microphotograph showing the state of
cancer in a cancer bearing mouse group which is not administer-
ed with GRA-M-l;
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38
Fig. 16 is a microphotograph showing the cancer cell
tissue of a cancer-bearing mouse group which is not administer-
ed with the GRA-M-l;
Fig. 17 is a microphotograph of the cancer cell
tissue of the group which is administered with GRA-M-l;
Fig. 18 shows an SDS gel electrophoresis diagram of
GRA using protein staining by C.B.B. method;
Figs. 19 to 21 show SDS gel electrophoresis diagrams
of GRA using sugar coloration by PAS method;
Fig. 22 is a schematical illustration of the results of SDS gel
electrophoresis diagram of GRA using pro~ein staining by C.B.B. method; and
Fig. 23 represents a graph showing tumor growth rate
in C3H/HQ mouse transplanted with X5563 immune mouse spleen
cells and X5563 cells.
DETAILED DESCRIPTION OF THE INVENTION
.
The killer cells of the invention can be prepared,
for example, by a method in which~GRA is used to sensitize
lymphocytes.
GRA as used in the above method can be obtained
from cancer cells containing GR~ of humans or animals, e.g.,
cultivated cancer cells, transplanted cancer cells, spon-
taneausly-occurring cancer cells, chemical substance or
virus-induced cancer cells, and cancer cells derived from
operated tissues, by the following procedure
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Cell membrane components are separated from cancer
cells as described above and treated with a lectin which
combines specifically with a terminal galactose or a terminal
N-acetylgalactosamine, whereby GRA is combined with the lectin and
can be easily separated.
Suitable examples of the galactose-binding lectin
include peanut lectin, Ricinus communis lectin and Soybean
(Glycine max) lectin (see J.B.C., 250, 8518-8523 (1975);
Biochem. Biophys. Res. Comm., 62, 14~ (1975); Z. Immunitaets-
forch, 138, 423-433 ~1969); Br. J. Exp. Pathol, 27, 228-236
~1946); Proc. Nath. Acad. Sci. USA, 75, No. 5, 2215-2219 (1978);
, _ _ _ _
Biochemistry, _, 196-204 (1974); and Carbohydrate Research,
51, 107-118 (1976)). Suitable examples of the N-acetyl-
galactosamine-binding lectin include Dolichos bean (Dolichos
biflorus) agglutinin, braid orange lectin, Helix pomatia
lectin, lima-bean (Phaseolus limensis) lectin, soybean
(Glycine max) lectin and Bauhinia bean (Bauhinia purpurea)
lectin.
The separation of the cancer cell membrane compo-
nents can be achieved by known techniques such as a homogeni-
zation method and a solubilization method using a dissolving
agent. It is more advantageous to employ a method in which
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cancer cells are homogenized in physiological saline or in
an appropriate buffer, a portion precipitated is collected
by a technique such as centrifugal separation and dissolved
in physiological saline or a buffer by the use of a dis-
solving agent, and the supernatant portion is separated bya technique such as centrifugal separation. Dissolving
agents which can be used include surface active agents which
are generally known to be capable of dissolving cell membrane,
such as nonionic surface active agents, e.g., TRITON X-100
~produced by Wako Pure Chemical Industries Ltd.) 3 NP-40
(produced by Shell Co., Ltd.), digitonin, and urea, and
anionic surface active agents, e.g., sodium dodecylsulfonate
(SDS).
From the thus-obtained cell membrane components
can be separated GRA capable of combining with lectin by
usual physical chemical or biochemical techniques utilizing
the properties of lectin. Examples of such techniques
include affinity chromatography utilizing a column carrier
containing lectin, an immune precipitation method using GRA
antibody or the like, a dialysis method, a gel filtration
method, an electrophoresis method, a physical precipitation
method Usillg a sugar protein-precipitating agent, e.g., poly-
ethylene glycol and acetone, and a combination thereof.
More pTeferred is affinity chromatography utilizing a column
carrie~ containing lectin, and the column carrier can be
~Z4~ ;3~
easily prepared by immobilization of lectin on an insoluble
support. Such immobilization of lectin on an insoluble
support can be performed by known techniques which are con-
ventionally employed in immobilization of biosubstances.
Of these techniques, it is preferred to use a cyan bromide
activation polysaccharide method and an immobilization me~hod
using N-hydroxysuccimide esters. The cyan bromide activation
polysaccharide method is a method in which an insoluble
support is treated with cyan bromide, and the activated
product thus obtained is subjected to a coupling reaction
with lectin under mild conditions to immobilize lectin. In
treating the insoluble support with cyan bromide, for example,
basic compounds such as sodium hydroxide and sodium hydrogen-
carbonate are used to adjust the pH to from 7 5 to 12, and
the support is treated in a solvent such as water, aceto-
nitrile, or a buffer maintained at pH 7.5 to 12, for example,
a 0.1 M sodium hydrogencarbonate buffer (pH about 8.7) and
0.01 M phosphate buffer (pH about 7.7), at room temperature
for about 1 ~o 12 minutes. Usually the amount of the cyan
bromide used is preferred to be equal to that of the insoluble
support.
Any known insoluble support which is o low non-
specific adsorption to all biosubstances, has high porosity,
contains a functional group capable of immobilizing bio-
substances under mild conditions~ and is chemically and
-.
1 physically sufficiently stable can be used in the invention.
Insoluble supports which can be used include a support
made of cellulose, e.g., aminoethyl cellulose, carboxy-
methyl cellulose, bromoacetyl cellulose, and p-anilino
cellulose, a support of cross-linked dextranr e.g.,
SEPHADEX* and CM-SEPHADEX* ~produced by Farmacia Corp.~,
and a support of agarose, e.g., SEPHAROSE* 2B, SEPHAROSE*
4B, and SEPHAROSE* 6B (producecL by Farmacia Corp.).
In the coupling reaction of the cyan bromide-
activated support as obtained above with lectin, the cyan
bromide-activated support is used in an amount from 30 to
80 times the lectin, and they are reacted in an appropri-
ate solvent such as a 0.1 mol/Q aqueous solution of sodium
hydrogencarbonate ~containing 0.5 mol/Q of sodium chloride;
pH: 8.4) at a temperature of from O to 40C, preferably
from 2 to 8C for a period of from about 10 to 20 hours.
In this way, there can be prepared a carrier for affinity
chromatography containing lectin.
By chromatography using the lectin-containing
carrier as prepared above, the desired GRA combines with
the lectin contained in the carrier and its trapped in
the column. Subsequently, substances capable of combin-
ing with e.g.,
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lectin can be passed through the column to perform an exchange
reaction, or alternatively an adsorption separator (eluting
solution), e.g., a high concentration salt, an aqueous solu-
tion of potassium thiocyanate, and a nitrate buffer, is passed
through the column to dissociate and obtain the desired GRA.
In the exchange reaction, examples of the substances
capable of combining with lectin when a carrier :For affinity
chromatography containing galactose-binding lectin is used
include those which can combine with a terminal galactose-
combining lectin, e.g., galactose, disaccharides containinga terminal galactose and oligosaccharides containing a termi-
nal galactose, and examples of the substances capable of
combining with lectin when a carrier for affinity chromato-
graphy containing N-acetylgalactosamine-binding lectin is
used include those which can combine with a terminal N-acetyl-
galactosamine-combining lectin, e.g., N-acetylgalactosamine,
disaccharides containing a terminal N-acetylgalactosamine and
oligosaccharides containing a terminal N-acetylgalactosamine.
The thus-obtained GRA contains glycoprotein contain-
ing a galactose and/or N-acetylgalactosamine terminus, glyco-
lipid and/or saccharide.
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9~
The ~RA thus prepared can be lyophilized, if desired
and further purified using ordinary separation techniques. For
example, those GRA preparations isolated with a galactose-
binding lectin can be subjected to a separation method using a
N-acetylgalactosamine-combining lectin, and those isolated
with a N-acetylgalactosamine-combining lectin can be treated
by a separation method using a galactose-binding lectin.
Lymphocytes as used herein are not cri~ical~ and
any lymphocytes of normal or cancer-bearing human being or
animals can be used. Examples include those lymphocytes
derived from peripheral blood, bone marrow, lymph node,
spleen, tonsils, and thymus. These lymphocytes are isolated
by physical or chemical techniques, a surface membrane method,
or the like.
The sensitization of lymphocytes with GRA is per-
formed by cultivating the lymphocytes on a culture medium
containing GRA for a period of from 1 to 10 days 7 preferably
from 2 to 7 days.
As culture media for use in the sensitization of
lymphoc~tes, there can be used various common nutrient media
which are conventionally used in such cell cultivation.
PreferTed examples include a RPMI 1640 medium, an Eagle MEI~
cul~ure, etc. with human serum, calf fetus serum ~FCS), calf
serum, horse serum or the li~e added thereto. The amount of
GRA being added to the culture is usually from 1 to 1,000
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ng/ml and preferably from 1 to 500 ng/ml, as calculated as
an amount of sugar, per lxlO6/ml lymphocyte.
The cultivation is performed by the common method,
for example, at a pH of about 7.2 and a temperature of about
37C.
The killer cells of the invention as prepared
above are substantially normal lymphocytes and have a
GRA-specific cell-combatting activity For example, GRA-l-
~T~ which is one of the killer cells of the invention, has
properties common to human peripheral blood T cells and
shows ~ cell-combatting activity which is specific to cancer
cells containing GRA, which is shown in the example as des-
clibed heleinater.
Typical examples of the novel killer cells of the
1~ invention aTe GRA-l-KT and GRA-M-l which are prepared in the
examples as described hereinafter. All the killer cells are available
from the appli~nt and their deposition in ATCC has been requested.
The killer cells of the invention can be multiplied
unlimitedly on the above-
d~scribed culture medium containing a T cell growth factor~TCGF, IL-2 ). In this case, selective cultivation of
cloning of ~he ~iller cell may be performed by the conven-
ti~nal ~ltra-dilution method These killer cells can be
store~ st~ly ov~r a long period o time, for example~ in
2~ liquid ni~rogen
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~2~
The GRA can be used singly as an active ingredient
and additionally, can be used in combination with other
antibacterial agents and cancer-inhibiting agents. Cancer-
inhibiting agents containing the GRA of the invention as an
active ingredient may be in any form as long as they are in
the condition that the GRA is contained in effective amounts
Usually the agent is administered intravenously, subcutaneously,
intradermally or intramuscularly as a solution, a suspension or as
an emulsion. In addition, it may be provided as a dry product
which can be made liquid by the addition of a suitable carrier
prior to the use thereof. These liquid agent may contain a
suspending agent, e.g., methyl cellulose, an emulsifying
agent, e.g., lecithin, preservatives, e.g., methyl-p-hydroxy
benzoate, a stabilizer which does not exert as such adverse
lS influences on the immuni~ing function of humans or animals,
- a buffer, and the like.
Aqueous carriers which can be used include physio-
logocal saline, and non-aqueous carriers which can be~used
include vegetable oil, e.g.~, sesame oil, mineral oil, e.~.,
paraffin, animal oil, e~g., squallene, and propylene glycol.
In addltion, for the purpose of immunological enhancement,
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. ._.. .. ~
~2~g~
appropriate adjuvants may be incorporated. Adjuvants which
can be used include Freund's complete adjuvants, saponin for
animals, and aluminum hydroxide for humans.
The anticancer agent of the invention can be
administered once or repeatedly over a long period of time
to a cancer patient for the treatment of cancer, or can be
administered to one who is liable to suffer from cancer for
the preven~ion of cancer.
Since LD50 (mouce, intraperitoneal) of GRA is at
least 500 mg/kg as calculated as an amount of sugar9 the
anticancer agen~ of the invention is of low toxicity and,
therefore, it can be administered within a wide range of
dosage. Although the concentration of GRA in the anticancer
agent of the invention is not critical, it is usually pre-
ferred to be from 0.001 to 100 ~g/ml as calculated as an
amount of sugar. With regard to the dose of the anticancer
agent, it is usually preferred that the agen~ is administered
in an amount of 0.001 to 1,000 ~g/kg/day, at the same time
or in several portions, although it varies depending on the
extent of disease,and the age and sex of the patient.
The thus-prepared anticancer agent containing the
killer cell as an active ingredient is preferably used as
an injectable solution in combination with carriers which
are used -in the preparation of such blood medicines.
Carriers as used herein are no~ critical, but carriers
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. ..
~0~
having a tonicity equal to that of blood are preferred. In
particular, physiological saline is preferred. In the
preparation of the agen~, it is pre~erred that the killer
cell is sufficiently washed with physiological saline or the
like to remove the above-described culture medium and, there-
after, it is floated in a carrier.
The concentration of the killer cell in the agent
is not limited specifically, but it is preferably from 105
to 10 per milliliter. When the killer cell is adminstered
intraperitoneally in a dose of 108 per mouce, no toxity is
observed. Although the dose of the anticancer agent of the
invention varies depending on the degree of disease, and the
age and sex of the patient, it is usually preferred that the
- agent is administered in a dose of 105 to 1012/kg/day, in
lS one pOTtiOn or in divided portions.
The following example, reference examples, experi-
ment examples, and comparative examples are given to illust-
rate the invention in greater detail although the invention
is not limited there*o.
REFERENCE EXAMPLE 1
Locality of GRA
~l)-A Preparation of FITC-labelled Lectin ~PNA-FITC)
Ten milligrams of peanut lectin ~PNA, produced by
EY Co.) was dissolved in 2 ml o-E a 0.01 M phosphate buffer
containing 0.85% NaCl CpH=7.2). In 1 ml of a 0.5 M hydrogen-
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~z~
carbonate buffer (pH=9.0) was dissolved 2 mg of FITC ~pro-
duced by Sigma Laboratories Inc.), and a 0.5 ml portion of
the resulting solution was added to the above prepared PNA
buffer. The mixture was then stirred at room temperature
for 2 hours and, thereafter, it was separated on Sephadex
G25 (10 mm x 300 mm, produced by Farmacia Corp.). The
initial peak was collected. FITC/PNA ratio=lØ
(l)-B Preparation of FITC-Labelled Lectin (DBA-FITC)
In the same manner as in (l)-A above DBA-FITC was
obtained using DBA produced by EY Co. FITC/DBA ratio=0.9.
(l)-C Soybean agglutinin FITC (FITC-SBA) is available from
EY Co. FITC/SBA ratio=1.4.
~2) Locality of GRA in Various Cancer Cells
~a) Human cultured cancer cells ~lx106) were washed
three times with a 0.05 M tris hydrochloric acid buffer
containing 0.85% NaCl ~pH=7.2) by a centrifugal procedure and
then 100 ~1 o-f PNA-FITC, DBA-FITC or SBA-FITC ~200 ~g/ml) as
prepared in ~l) above was added thereto. The resulting mix-
ture was allowed to stand a~ room temperature for 30 minutes
to cause them to react. After the reaction was completed,
the reaction mixture was washed three times with a 0.01 M
phosphate buffer containing 0.85% NaCl ~pH=7.2) and, there-
after, the cells were placed on a glass plate and examined
under a fluorescent microscope.
Mouse X5563 and mouse MH134 were treated and
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.... - ,,, ''' ! '. : : " ' '
e~amined in the same manner as above.
The results are shown in Table 1. The cancer cells
used are all known and have been obtained from First Pathology
Laboratories, Medical Department of Niigata University.
Table 1
Positive Ratio of
Cancer Cells GRA _ (%)
PNA DBA SBA
Raji (Burkitt lymphoma) 98.3 1.4
Dauji ~Burkitt lymphoma) 93.1 5.2
BT-l (Burkitt lymphoma) 50.1 0
P-12 (T-cell lymphoma) 44.3 6.7
MOLT ~T-cell leukemia) 0.6 4.8
Fujimaki (B-cell lymphoma) 19.1 5.3
Oda ~IgD myeloma) 0.6 10.0
QG-56 (lung cancer, squamous) 70.4 2.0
PC-l (lung cancer, squamous) 78.4 0.4
PC-3 (lung cancer, adenocarcinoma) 77.1 0
QG-90 ~lung cancer, small cell)68.0 0
PC-13 (lung cancer, large cell)17.0 0
MK-2 (stomach cancer, poor
diferentiation) 63.7 0.1
KATO-III ~stomach cancer, signe~
ring cancer) 57 3 0
MKN-45 (stomach cancer, poor
differentiatlon) 1.0 40.3
MKN-l (stomach cancer,
adenocarcinoma-squamous) 4.6 0.4
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Positive Ratio of
Cancer Cells GRA (%)
-
PNA DBA SBA
MKN-28 ~stomach cancer) 0.4 0.1
MKN-74 (stomach cancer) 0.5 0
MGH-Ul (urinary bladder cancer) 37.4 0
KU-2 ~urinary bladder cancer) - 4.5 21.4
T-24 (urinary bladder cancer) 14.6 0
NBT-2 (urinary bladder cancer) 13.1 1.0
NRC-12 (renal cancer) 23.9 0
KU-l (renal cancer) 3.3 0.6
Kuramochi (ovarian cancer) 80.0 0
NB-l ~neuroblastoma) 50.9 1.7
YT-nu tneuroblastoma) 3.6 0.5
TGW-nu-l ~neuroblastoma) 4.1 0
TGW-nu-ll ~neuroblastoma) 2.0 1.0
GOTO (neuroblastoma) 0.5 0
ITO (embryonal carcinoma) 96.9 12.3
NEC-8 ~embryonal carcinoma) 44.6 0
SCH tchoriocarcinoma, stomach) 14.6 3.1
GCH ~chriocarcinoma, u*erus) 5.4 0
YN-l (rhabdomyosarcoma) 5.7 1.7
Mouse X5563 ~plasmacytoma) 92.0 0 90.6
Mouse MH134 ~hepatoma) 18.6 0 6.4
.. , . . - ~;
- ^ ... . ... . . .
(b) The ~alignant tissues from cancer patients were
passed through a stainless steel mesh (#150) to make single
cell suspension. After wasiling twice with 0.01M Tris-~lCl
buffer (pH=7.4~ containing 2mM CaC12, 2mM MgC12 and 0.85%
NaCl~ 5x105 cells were resuspended in 100 ~1 of the buffer.
One hundred ~1 of FITC-PNA or FITC-DBA (200 ~g/ml) was added
to the cell suspension and the mixture was incubated at room
temperature for 20 minutes. After washing three times with
cold PBS, the cell were observed under a fluorescene micro-
scope.
The results are shown in Table 2. The malignant
tissues from cancer patients have been obtained from Kansai
Medical University.
Table 2
GRA
No. Cancer Patient Tissue PNA DBA
1 Stomach cancer + +
2 Stomach cancer + +
3 Stomach cancer - -
4 Stomach cancer +
Stomach cancer + +
6 Stomach cancer +
7 Breast cancer ~ - -
8 Breast cancer +
9 Breast cancer +
Breast cancer +
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GRA
No. Cancer Pa~ient Tissue PNA DBA
11 Colon cancer ~ +
12 Colon cancer -
13 Esophagus cancer ~ -
14 Hepatoma - +
Note: Symbol "+" indicates GRA is expressed on the
cell surface.
Symbol "-" indica-tes GRA is not expressed on the
~ell surface.
REFERENCE EXAMPLE 2
Preparation o-f GRA
(l)-A Preparation of Immobilized Lectin ~PNA-Sepharose)
Three grams of CNBr-activated Sepharose 4B (pro-
duced by Farmacla Corp.) was fully washed with 1 mM HCl and
suspended in 200 ml of 0.1 M sodium hydrogencarbonate ~pH=
8.5). Then, 5 ml of a 0.01 M phosphate buffer ~pH=8.5)
containing 20 mg of PNA was added, and they were reacted at
25C for 2 hours while sometimes stirring to prepare PNA-
Sepharose.
~l)-B In the same manner as in ~l)-A above except that DBA
is used instead of PNA, DBA-Sepharose was obtained.
~2) Prepara-tion of GRA
~ a) BT-l (Burkitt lymphoma) cells (1.3x108) were washed
three times with physiological saline, and 30 ml of a 0.01 M
tris hydrochloric acid buffer (pH=7.4) containing 2% of
,
. .
1 "TRITON* X-100" (produced by Wako Pure Chemical Industries
Ltd.), 0.85% of NaCl, 2 m~l of CaC12, and 2 mM of MgC12 was
added thereto. The mixture was stirred at 4C Eor 15
minutes and then was subjected -to ultracentriEugal separ-
ation at a rate of 100,000 x g. O~ 28 ml of the thus-
obtained supernatent liquid, a 14 ml p~rtion was passed
through a column (diameter 0.5, length 1 cm) for affinity
chromatography, packed with PNA-agarose beads (produced
by Maruzen Co., Ltd.) which had been equilibrated by a
tris hydrochloric acid buffer (pH-7.4) containing 0.1%
TRITO~* X-100, 0.85% NaCl, 2 mM CaC12, and 2 mM MgC12.
After being washed with the same buEfer as used above,
it was eluted with a 0.01 M -tris hydrochloric acid bu~fer
(pH--7.4) containing 0.1 ~ lactose, 0.85~ NaCl, 2 mM CaC12,
2 mM MgC12, and 0.1% ~RITON* X-100. The thus-eluted
portion was dialyzed with a 0.01 M tris hydrochlorie
acid buffer containing 0.85% NaCl, 2 mM MgC12, and 2 mM
CaC12 for 48 hours to obtain 17 ml of a GRA solution.
With the GRA solution, the amount of protein and the
amount of sugar were measured by the Folin-Lowry method and
the phenol-sulfuric acid method, respectively, and they
were found to be 544 ~g and 120 ~g, respeetively, This is
hereinafter referred to as "GRA-l".
~b) C3H/He Mouse mammary tumor (MMT) cells
(lx101~ were washed three times with physiologieal saline,
and 20 ml of a 0.01 ~ tris hydrochlorie acid buffer
(pH-7.4) c~ntaining
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2% TRITON X-100, 0.85~ NaCl, 2 m~l CaC12, and 2 mM MgC12 was
added thereto. The mixture was stirred at 4C for 30 minutes.
Subsequently the mixture was subjected to ultracentrifugal
separation at a rate of 100,000 x g for 2 hours, and the
supernatant liquid was dialyzed over-night with a 0.1 M tris
hydrochloric acid buffer (pll=7.4) containing 0.85Po NaCl, 2
mM CaC12, and 2 mM MgC12. The thus-dialyzed liquid was
concentrated to 3 ml, and a 1 ml portion was then passed
through a column ~diame-ter0.5 ~l, length 2 cm) for affinity
chromatography, packed with the same PNA Sepharose as used
above which had been-equilibrated with a tris hydrochloric
acid buffer ~pH=7.4) containing 0.005% TRITON X-100, 0.85%
NaCl 3 2 mM CaC12, and 2 mM MgC12. After being fully washed
with the same buffer as used above, it was eluted with a
0.01 M tris hydrochloric acid buffer ~pH=7.4~ containing
0.1 M lac-tose, 0.85% NaCl, 2 mM CaC12, 2 mM MgC12, and 0.005%
TRITON X-100, and the thus-eluted portion was dialyzed for
4~ hours with a 0.01 M tris hydrochloric acid buffer ~pH=7.4)
containing 0.85% NaCl, 2 mM CaC12 and 2 mM MgC12 to obtain
2 ml of a GRA solution. With the GRA solution thus obtained,
the amount of protein and the amount of sugar were 156 ~g
and 94 ~g~ respectively. This is hereinafter referred to
as 1tGRA-M-l"
- 22 -
(c) Approximately 120 g (wet weight) of KATO-III was
a homogeni7ed in 100 ml of PBS by a Waring blender. After `
centrifugation at 100,000 g for 1 hour, the pellet was dis-
solved with a in 100 ml of 2% TRITON X-100 in 0.01 M Tris-
HCl buffer (pH=7.6) containing 0.15 M NaCl. The supernatantcollected by centrifugation at 100,000 g for 1 hour and
applied to a column of PNA-Sepharose 4B (0.8x15 cm) equili-
brated with 0.015% TRITON X-100 in 0.01 M Tris-HCl (pH=7.6)
containing 0.15 M NaCl. After washing with 50 ml of the
buffer, the GRA were eluted with the buffer containing 0.1
M lactose. The eluted GRA dialysed against 0.85~ NaCl,
concantrated by Sephadex Pharmacia Co. and stocked at -20C
before use.
The amounts of protein and sugar determined in the
same manner as in (a) above were found to be 2.0 mg and 0.8
mg, respectively. This is hereinafter referred to as "GRA-2"
(d) In the s~me manner as in (c) above~ the ~ollowing
GRA samples were obtained.
23 -
Table 3
GRA
GRA Material Protein Sugar
Sample SourceAmount Content Content
~g~ mg) (mg)
GRA-3 BT-l 33 0.5 0.09
GRA-4 Breast Ca. 5 0.24 0.5
~excised)
GRA-5 QG-56 24 0.6 0.38
GRA-6 QG-90 26 1.0 0.54
GRA-7 Raji 29 0.78 0.45
GRA-M-2 MMT 200 11.3 28.4
GRA-M-3 LLC 14.4 0.06 0.07
GRA-M-4 MH-134 85 0.65 0.35
GRA-M-5 X-5563 25 0.56 0.23
~e) In the same manner as in ~c) above e~cept that MKN-45
~about 2g g) was used instead of Kato-III and DBA-Sepharose
obtained in ~l)-B above was used instead of PNA-Sepharose 4B
and elution was carried out with N-acetylgalactosamine, a
GRA preparation having a protein content of 0.03 mg and a
sugar content of 0.01 mg was obtained. This is hereinafter
referred to as "GRA-8".
~f) GRA-3 prepared in ~d) above ~5 mQ) was charged in DBA-
Sepharose column and eluted with Tris-HCQ buffer (0.015%
Triton X-100, 2 mM MgCQ2 CaCQ2, O.85% NaCQ) to obtain 4 mQ
fractions Nos. 1-12. Fractions Nos. 1-3 are termed "GRA-3-A"
and Nos. 4-12 "GRA-3-B". Then, the column was eluted with
the same buffer but containing 0.1 M N-acetylgalactosamine
- 24 -
9~
to obtain fractions, which is termed "GRA-3-C".
~g) SDS Gel Electrophoresis
Each of the GRA preparations according to the above
procedures was subjected to electrophoresis according to the
method described in Fairbanks et al: Biochemistry vol. 10
p.2606, 1971.
The results obtained are shown in Figs. 18 to 22.
In Figs. 18-19~ numerals 1 to 5 indicate the following.
1 --- standard; 2 --- GRA-~1-3; 3 --- GRA-7;
4 --- GRA-l; and 5 --- GRA-2
In Fig. 20 numerals 1 to 4 indicate the following.
1 --- standard; 2 --- GRA-M-2; 3 --- GRA-6;
4 --- GRA-5
In Fig. 21 numerals 1 to 3 indicate the following.
1 --- GRA-M-4; 2 --- GRA-M-5; 3 --- standard
In Fig. 22 numerals 1 to 4 indicate the following.
1 --- GRA-3; 2 --- GRA-3-A, 3 --- GRA-3-B;
4 --- GRA-3-C
Fig. I8 shows the state of GRA s~bjected to
protein staining reaction according to C.B.B. method
~Fairbanks et al: Biochemistry vol. 10 p.2606, 1971).
Figs. 19-21 shows the state of GRA subjected to
sugar coIor reaction according to PAS method (R.M. Zacharius
et al: nal. Biochem. vol. 30 p.l48 ~1962)).
Fig. 22 shows schematical illustration of the results
of staining according to the C.B.B. method described above.
- 25 -
For the standards substances listed belol~ from
Biorad Lab. Calif. U.S.A. were used.
200 K dalton; myosin
116 " ; ~-galactosidase
92.5 " ; phosphorylase
66.2 " ; BSA
45 " ; ovalbumin
21.5 " ; soybean trypsin inhibitor
REFERENCE EXAMPLE 3
Preparation of TCGF
(a) The spleen (4 kg) of Japanese monkey (obtained from
Japan Plymates Co., Ltd.) was excised and washed twice with
a RPMI-1640 culture medium ~produced by Flow Laboratory Co.
Ltd.). Cells were filtered by the use of Mesh ~produced by
Milipore Inc., 150 mesh) and were subjected to a specific
gravity centrifugal method (specific gravity, 1.076) to
obtain 2 liters of 2xlO9/ml of lymphocytes. The thus-
obtained lymphocytes were washed three times with a RPMI-1640
- culture medium~ and the lymp~hocyte number was adjusted to
5x107 per millillter by the use of the same medium as used
above, containing 10% FCS. It was then allowed to stand in
a carbon dioxide fermentation apparatus at 37C for 1 hour.
26 -
~2ai1~
The supernatant lymphocytes were recovered and the lymphocyte
number was adjusted to lx106 per milliliter by the use of the
same culture medium as used above, containing 1% FCS. Sub-
sequently 1 ~g/ml of indomethacin ~produced by Sigma
Laboratories, Inc.) and 0.2% PHA-P ~produced by Difco Co.)
were added, and they were cultured in a carbon dioxide gas
fermentation apparatus at 37C for 48 hours. A centrifugal
separation procedure (3,000 rpm) was applied for 10 minutes,
and the resul~ing supernatan-~ liquid was recovered and
sterilyzed by filtering with a Millipore filter ~produced by
Millipore Inc., 0.2 ~m) to obtain 2 liters of ~CGF.
~ b) The source of TCGF was the supernatant of mix-
cultured peripheral blood lymphocytes of 10 healthy donors.
Non-adherent lymphocytes prepared from C.F ~Conray. Ficol
~Japan Immunoresearch Co.)) separated lymphocytes by adsorp-
tion on plastic surface at 37C for 1 hour were suspended in
RPMI 1640 medium containing 1% FCS ~l.Sx106 cells/ml) and
incubated with 0.2~ PHA-P, indomethacin (1 ~g/ml) and human
~-cell line ~T-l) ~1 sx105 cells/ml) pretreated with mito-
mycin C (50 ~g/ml). After 48 hours, the culture supernatan~was harvested and used as TCGF source. ~H. Inoue et al,
Scand. J. Immunol. 12 p.149-154 (1980))
- 27 -
9~
REFERENGE EXA~IPLE 4
Preparation of Lymphocytes
(1) Human Peripheral Blood Lymphocytes
Blood ~50 ml) obtained ~rom a healhty adult or various
cancer patients by heparinization was subjected to centrifugal
separa~ion by ~he use of Ficol Pack ~produced by Farmacia Japan
Co., Ltd.) to obtain 5x107 of peripheral blood lymphocytes.
~2) Mouce Spleen Lymphocytes
The spleen of a C3H/He mouce ~male, 6w) was excised
and washed twice with a RPMI-16~0 culture medium. The spleen
was then loosened by a syringe needle and passed through a
stainless steel screen (100 mesh) to remove large pieces.
The thus-filtered cells were washed twice with the same
culture medium as used above and was subjected to centrifugal
separation at a rate of 1200 rpm for 10 minutes to obtain
4xlO o spleen lymphocytes.
EXAMPLE 1
-
GRA-l (amount of protein: 40 ~g/ml; amount of
sugar: 7.5 ~g/ml) as obtained in Reference Example 2 ~2)-
~a)) was diluted to 1,000 times the original volume with aRPMI-1640 culture medium containing 15 ~ FCS to prepare a
sensitization culture medium.
Human periphe~al blood lymphocytes ~5X106/5 ml) as
obtained in Reference Example 4 (~1)) was added to 5 ml o
~he sensitization culture medium as prepared above which
- 28
9~
was placed on a laboratory dish, and cul~ivated at 37C for
2 days They were further cultivated on the RP~ 1640 cul-
ture medium containing 20% TCGF and 15% FCS, as obtained in
Reference Example 3, for additional five days to obtain 20
ml of a killer cell solution containing 1x106 killer cells
per milliliter. This is hereinafter referred to as "GRA-l-
K-T".
EXAMPLE 2
The mouce spleen lymphocytes as obtained in Refer-
ence Example 4 (~2)) were adjusted to a number of 5xlO6/ml by
the use of a RPMI-1640 culture medium containing 15% FCS.
Then, GRA-M-l as obtained in Reference Example 2 ~(2)-~b))
was added ~o that the final amounts of protein and sugar
were 1.5 ~g/ml and 0.9 ~g/ml, respectively A 5 ml portion
I5 of the resulting mixture was cultivated on a laboratory dish
(60 mm x 15 mm, produced by Falcon Co.) at 37C for two days.
The formation of cloning was observed. The portion was fur-
ther cultivated Oll a RPMI-1640 culture medium having 15% FCS
containing 20% by volume of TCG~ ~produced by Japan Immuno
Research Laboratories Co., Ltd.) for additional 4 days to
obtain 50 ml of a killer cell solution containing 1x106 killer
cells per milliliter. This is hereinafter referred to as
"GRA-M-l-K-T".
- 29 -
EXAMPLE 3
Peripheral blood lymphocytes (5x106) of healthy donor was
incubated in RPMI-1640 medium containing 50 ng/mQ (protein
content) of GRA-2 and 15% FCS at 37C. On day 2 the human
TCGF described above was added to the medium until the con-
centration reached Z0% and incubation was continued for fur-
ther 3 days ~o obtain killer cells. This is hereinafter
referred to as "GRA-2-K-T".
EXAMPLE 4
Killer cell preparations GRA-8-K-T, GRA-3-A-~-T and
GRA-3-C-K-T were prepared in the same manner as in EXAMPLE 1
using GRA-8, GRA-3-A and GRA-3-C, respectively (protein
content: 50 ng/mQ) and the TCGF obtained in REFERENCE EXAMPLE
3-~b).
EXAMPLE 5
C3H/He mice ~female; 8 weeks) was implanted intradermally
with X5563 cells ~106) of the same strain and after 7
days the tumor was excised surgically. After another 7 days
X5563 cells ~105) o the same strain was inoculated~
and mice resistant to inocula~ion were named immune mouse.
The spleen cells of the immune mice and C3H/He normal
mice were prepared according to commonly used method.
Each lot of spleen cells ~5xl06/well) was sensitized
with 40 ng/mQ (protein content~ of GRA-M-S in RPMI-1640 medium
containing 15% FCS for 5 days to obtain killer cells.
- Killer cell preparation obtained from normal spleen cells
~ 30 -
is hereinaf-ter referred to "GRA-M-5-K-T-l" and that obtained
from immune spleen cells is hereinafter referred to "GRA-M-5-
K-T-2".
EXAMPLE 6
Human peripheral blood lymphocytes ~5x106) were incubated
in S mQ of RPMI-1640 medium containing 50 ng/mQ (protein
content) of GRA-l at 37C for 2 days. On day 3, the lympho-
cytes were transferred to RPMI-1640 medium containing 10%
serum from the donor of the lymphocytes and 0 to 100 ng/mQ
~protein content) of GRA-l and incubation was continued for
further 5 days to obtain killer cell preparations shown in
Table 4 below.
Table 4
GRA Concentration ~n~/mQ)
15Initial Day Day 3 Killer Cell
0 GRA-l-K-T-l
1.6 GRA-l-K-T-2
3.2 GRA-l-K-T-3
6 GRA-l-K-T-4
12.5 GRA-l-K-T-5
GRA-l-K-T-6
GRA-l-K-T-7
100 GRA-l-K-T-8
EXAMPLE 7
(a) Peripheral blood lymphocy~es ~PBL) from various cancer
- patients after operation were sensitized with GRA-3 to obtain
- 31
killer cell perparations. PBL (5x106) from the patients were
incubated in RP~ 1640 medium containing 50 ng/mQ (protein
content) of GRA-3 for 2 days and then in RP~II-1640 medium
containing 20% TCGF and 15% FCS for further 5 days to obtain
killer cells shown in Table 5.
Table 5
Peripheral Blood Lymphocytes
Days after
Cancer P-GRA D-GRA Operation Killer Cell
Gastric ca. ~ -~ 14 GRA-3-K-T-l
" + + 35 GRA-3-K-T-2
Breast ca. ~ - 21 GRA-3~K-T~3
" ~ - 7 GRA-3-K-T-4
" + - 35 GRA-3-K-T-5
Gastric ca. + - 21 GRA-3-K-T-6
In the above table, P-GRA and D-GRA indicate locality
of GRA expressed on the malignant tissue (excised by opera-
tion) from the cancer patients from whom PBL was collected as
determined in the same manner as in REFERENCE EXA~PLE 1, 2-
~b). P-GRA and D-GRA are results obtained using ~ITC-PNA
and FITC-DBA9 respectively, The days after operation indicate
the timing when PBL was collected after the operation.
tb) PBL (5x106) collected from breast cancer patients after
21 days from the operation was incubated in RPMI-1640 medium
containing 50 ng/mQ (protein content) of GRA-3 and 10% serum
from tlle patient for 7 days to sensitize PBL and obtain killer
cell preparation. This is hereinafter referred to "GRA-3-K-T-7".
- 32 -
EXA~IPLE 8
GRA l-K-T (108) as obtained in Example 1 was dis-
solved in 10 ml of physiological saline to prepare an injec-
table solution.
EXAMPLE 9
(i) GRA-M-l as obtained in Reference Example 2 ((2)-(b))
was diluted with physiological saline so that the amounts of
sugar and protein be 1 0 ~g/ml and 1.6 ~g/ml, respectively,
to thereby prepare an anticancer agen~ No. 1.
~ii) A tumor lump of C3H/He spontaneously occurring breast
cancer was sterilely cut to 5 mm cube clump and transplanted
to under the back skin of each o ten C3H/He mice of the same
strain as above (7-week-age, males). Seven days after the
transplantation, the fixation and multiplication of the tumor
were confirmed. To five o the mice were each administered
subcutaneously the anticancer No. 1 as prepared in (i) above
in a dosage of 300 ~l per day at two day intervals. The
remaining five mice were used as untreated controls. Ten days
after the first administra~ion~ the tumor was excised by ope-
ration~ and the mean weigh~ was measuredO At the same time,
pathohistological examination was performed.
Tumor Yolume:
Adminstered group 22.3 mm3 (Fig. 14)
Control 162.7 mm3 (Fig. 15)
This means that there was a 86.3% reduc~ion in the tumor.
Pathohistological Examination:
~z 7
- Ja -
9~
In the control group (Fig.16), birds' nest bodies
of cancer were formed, the type o:E tissue was like medullary
canalicular cancer and the mul~ip:Lication of tumor cells was
observed all over the tissue. On the other hand, in the
agent administered group ~Fig.17), the cancer cells caused
liquefaction necrosis at the sites where the cancer cells
were formed, and calcification and fibrosis occurred,
leaving only a very limited amount of cancer cells. Thus,
-the anti-tumor proper~ies of the anticancer agent of the
invention were observed.
T~ST EX~VIPLE 1
GRA-l-K-T (1 ~1~ as obtained in Example 1 was
placed on a microplate (produced by Falcon Corp.) and was
allowed to stand at room temperature for 15 minutes. Then,
4 ~1 of FCS (produced by Falcon Corp.) was added and the
mixture was allowed to stand at room temperature for 30
minutes. Neuraminidase-treated sheep red blood cells (SRBCN)
adjusted in number to lx109 per milliliter and 5 ~1 of a
0.01 M phosphate bu~er (pH=7.2) ~ith 0.~5% NaCl added were
added, and the plate was subjec~ed ~o a centrifugal separa-
tion procedure at a rate of 600 rpm for 5 minutes. The plate
was then reversed, and unreacted SRBCN was rcmoved. A dye-
ing solution (Brilliant Cresyl Blue, produced by Merck ~ Co )
was added ~o dye the lymphocytes, and rosette-forming posi-
tivity was examined. As a result, it has been found that
- 34 -
at least 98% exhibits rosette-forrning positivity (T-cells.)
TEST EXA~IPLE 2
-
Specific Cancer Cell-Killin~ ActivitY
~a) Of the cells shown in Table 1, the following five
cell strains having different GRA positivity ratios were
used as targe-t human cancer cells.
Targe-t Cancer Cells:
- Mo. 1 BT-l (Burkitt lymphoma)
No. 2 Daudi (Burkitt lymphoma)
No. 3 KATO-III (stomach cancer)
No. 4 MKN-45 (stomach cancer)
No. 5 MOLT ~T cell leukemia)
On a microplate ~produced by Falcon Corp.) were
laminated 5x104 per well of target cancer cells by a centri-
fugal proceduTe at a rate of 800 rpm for 5 minutes. Then,
4x10 per well of GRA-l-K-T as obtained in Example 1 was
gently added and incubated for 1 hour.
The killing activity was determined according
to the degree of plaques-formation, and was rated as follows:
~+ Killing activity is significantly observed.
-~ ICilling activity is observed.
Killing activity is slightly observed.
- Killing activity is not observed.
In a control group~ there were used unsensitized
human peripheral blood lymphocytes which had been prepared
- 35 -
in the same manner as in Example 1 except that GRA was not
used. The results are shown in Table 6.
It is apparent from Table 6 that the killer T cells
produced by the process of the invention have strong GRA-
specific cytotoxic activity.
Table 6
_._
Target Cancer GRA Positivity Determination of
Cell Ratio ~%) Plaaue-Formation
GRA-l-IC-T Daudi (Fig. 1) 93.1 ~-~ (Fig. 2)
Group
KATO-III (Fig. 3)57.3 + (Fig. 4)
BT-l (Fig. 5) 50.1 ++ (Fig. 6)
MKN-45 ~Fig. 7) 1.0 + (Fig. 8)
MOLT (Fig, 9) 0.6 (Fig. 10)
Control ~T-l 50.1 - (Fig. 11)
Group
(b) The same -~arge~ cancer cells as used in (a) above
(3.2x106) were mixed with 8x105 GRA-l-K-T (cell ratio: 5/1)
and the resulting cell mixture (total number: 4x106) was
cultivated on a RPMI-1640 medium containiJlg 15% FCS. After
one hour, the number of remaining cells was counted and the
% cytotoxicity was calculated by the following equation.
number of cells after
% Cytotoxicity = (1 ~ (number of cells before) x 100)
cultivation (4x106)
The -results are shown in Table 7
- 36 -
9~3
Table 7
Number of Cells
Before Culti- After Cul~i-
Target Cell vation vation _ % Cytotoxicity
Daudi 4X106 2.9x106 28
KATO-III 4X106 3.7x106 7.5
BT-l 4xlo6 3,2x106 20
MKN-45 4X106 3.9x106 2,5
MOLT 4X106 4.2x106 -5
~c) The procedure of (b) above was repeated with the
exception that the mixing ratio of GRA-l-K-T to target cancer
cell was changed to 5/3. The results are shown in Table 8.
Table ~
Number of Cells
Before Culti- After Culti-
Target Cell vation _ vation ~ Cytotoxicity
Daudi 4xlo6 3.6x105 91
KATO-III 4xlo6 3~6X106 24
BT-l 4xlq6 1.4x106 65
M~N-45 4X106 3.7x10 7.2
MOLT 4X106 4.1x106 -3
In the above test it is observed that GRA-l-K-T shows a high
binding activity to Daudi, ICATO-III and BT-l but only a low
binding activity to MKN-45 and MOLT.
TEST EXAMPLE 3
C3H/He spontaneously-occurring breast cancer-bearing
mice were administeTed subcutaneously by GRA-M-l-K-T as obtained
- 37 -
in Example 2 at a dosage of 3x106/0.3 ml/mouse three times per
week every other day. After ten days, the focus was taken out
and examined.
As shown in Fig. 129 the infiltration of lymphocytes
into cancer cells occurred, and the breakage of the tumor area -
was observed. Also, from Fig. 13, it has been observed that the
calcification of the tumor area occurred, and thus it can beseen
that the killer cells of the invention have antitumor activity.
COMPARATIVE EXAMPLE 1
In this example, cancer cells per se were used as specific
antigens in place of GRA for use in the process o:E the invention.
- Cancer cell-sensitized lymphocytes were obtained in
the same manner as in Example 1 except that, in place of ~RA9
BT-l, Daudi~ KATO-III, or MKN-45 was used at a level of lx105
per laboratory dish.
~ ith these lymphocytes9 the cytotoxic activity was
examined in the same manner as in Test Example 2 ~(a)). The
results are shown in Table 9.
It c2n be seen from Table 9 that the above-prepared
lymphocytes do not have any cytotoxic activity.
Table 9
Cells for Use in Sensiti-
zation of Lymphocytes
Target Cell BT-l Daudi KAT0-III MKN-45
BT-l
Daud~
KATO-III
MKN-45
TEST EXAMPLE 4
In the same manner as in TEST E~A~IPLE 2-(b), the cancer cell-
killing activity of GRA-8-lC-T, GRA-3-A-K-T and GRA-3-C-K-T
obtained in EXAMPLE 4 was determined. The results obtained
are shown in Table 10.
Table 10
Target % Cytotoxicit
Cell GRA-8-K-T GRA-3-A-K-T GRA-3-C-K-T
KATO-III 8.0 5.0 5.0
BT-l 4.3 5.0 4.0
MKN-45 20 5.0 20
MOLT 0 0 0
TEST EXAMPLE 5
~a) Cytotoxicity of each of killer cell preparations obtained
in EXAMPLE 6 was determined by 51Cr release test (J. Immunol.
122, 2527-2533 (1979 ). That is, 50 ~Ci of radioactive 5lCr
(Japan Isotope Association) was added to XATO-III (2x107) and
the cells were incubated at 37C for l hour in RPMI-1640
medium and sufficiently washed by cen~rifugation to obtain
51Cr-labeled target cells. Killer cells ~effector cells)
~2x106~ were added to the target cells (lx105) (thus E/T =
20/1) and the mixture was incubated at 37C for 4 hours in
RPMI-164Q medium. Supernatant was collected by centrifuga-
tion and its radioactivity was determined by liquid scintilla-
tion coun~eT.
- 39 -
~2~
The specific 51Cr Release (%) which corresponds to the
cytolytic activity of effector cells was calculated according
to the following equation.
Specific 51Cr Release (%)
= ~Release in Test) - (Spontaneous Release) x 100
(Maximum Release) - (Spontaneous Release)
~The maximum release indicates the radioactivity when all the
cells are lyse~),
The results obtained are shown in Table 11.
Table 11
Specific 51Cr
Killer Cell Release (%)
GRA-l-K-T-l 13
GRA-l-K-T-2 25
GRA-l-K-T-3 22
GRA-l-K-T-4 20
GRA-l-K-T-5 22
GRA-l-K-T-6 25
GRA-l-K-T-7 27
GRA-l-K-T-8 32
From the results shown in Table 11 above, it can be
seen that TCGF an~ FCS have no relation wi~h the induction
of killer cells.
(b) Cytotoxicity of GRA-2-K-T obtained in EXAMPLE 3 was
de~ermined by 51Cr release ~est in the same manner as in (a)
above. Speci~ic 51Cr release of 14.3~ was observed on 51Cr-
labeled KATO-III as target cell ~E/T = 20/1).
- 40 -
TEST EXAMPLE 6
(a) Using ICATO-III (E/T = 20/1) as target cell cytotoxicity
of killer cells obtained in EXAMPLE 7-(a) was determined in
the same manner as in TEST EXAMPl,E 2-(b),
The results obtained are shown in Table 12 below.
Table 12
Killer Cell % Cytotoxicity
GRA-3-K-T-1 38.0
GRA-3-K-T-2 18.2
GRA-3-K-T-3 20.9
GRA-3-K-T-4 23.2
GRA-3-K-T-5 24.5
GRA-3-K-T-6 20.2
(b) Cytotoxicity of GRA-3-K-T-7 obtained in EXAMPLE 7-(b)
was determined using 51Cr-KATO-III ~E/T = 20/1) as target
cell in the same manner as in TEST EXAMPLE 5. Specific 51Cr
release of the killer cell was found to be 25.5%.
TEST EXAMPLE 7
Cytotoxici~y of ~he killer cells ob~ained in EXAMPLE 5
was determined by 51Cr release test in the same manner as in
TEST EXAMPLE 5. The target cell used was 51Cr-labe]ed X5563
cell. As a control lymphocytes obtained in the same manner
as in EXAMPLE 5 except that sensitization of spleen cells was carried
out with lxlO5/well of mitomycin C-treated X5563 cells (5xlO6/mQ
of X5563 cells were treated with 50 ~g/mQ of Mitomycin C for
60 minutes) ins~ead of GRA-M-5 were used.
- 41 -
The results obtained are shown in Table 13 below.
Table 13
Specific 5lCr Release (%)
E/T ratio
Killer Cell 40:1 20:1 10:1
GRA-M-5-K-T-1 12.7 6.3 5.7
GRA-M-5-K-T-2 18.4 20.6 13.7
Control 0.0 0.0 0.0
TEST EXAMPLE 8 ~H-2 Assay)
GRA-M-l, GRA-~ 3 and GRA-M-4 obtained in REFERENCE
EXAMPLF.2 above were serially diluted with PBS (0.85% NaCQ)
to prepare samples.
Anti-H-2 serum rom National Institute of Genetic
Research and the above sample were mixed and incubated at 4C
for 2 hours and a target cell corresponding to the anti-H-2
serum used was added thereto. Spleen cell or lymph node cell
obtained -from B10 ~H-2b) and BlO-BR (H-2k) mice by conven-
tional method were used as target cell. After washing the
cells with PBS, complement (rabbit) was added to the cell and
the cells were incubated a~ 37C for 1 hour and stained with
0.2% trypan blue-PBS to determine % cytotoxicity. The anti-
H-2 serum was used in a maximum dilution such that it showed
at least 95% cytotoxicity in the absence of GRA.
The blocking effect by GRA was determined for systems
shown in Table 14 below.
- 4~ -
able 14
GRA Anti-H-2 Serum, Concentration Target Cells
.. . . .
GRA-M-l D-23 ~anti-H-2Kk), X80 BlOBR (H-2}C~
or k spleen cells
D-32 (anti-~-2D ), X300
GRA-M-3 D-33 (anti-H-2Kb),X600 B10 (H-2b)
or b spleen cells
D-2 ~anti-H-2D ),X80
GRA-M-4 D-23 ,X80 BlO-BR ~h-2k)
or lymph node cells
D-32 ~X300
The results obtained are shown in Table 15.
Table 15
% Cytotoxicity
. _
Anti-H-2 _ Dilution of Sample __
GRASerum XZ X4 X8 X16 X3Z X64 X128 X256 X512 0 *
I - - 13 14 12 13 14 13 14 13 14 13
D-23 - 97 99 96 97 97 96 97 96 96 14
D-32 - 95 95 -95 95 94 95 95 96 95 13
II - - 19 15 14 12 15 12 11 12 13 14
D-33 - 98 98 99 99 98 99 99 99 99 15
D-2 - 95 96 95 95 97 95 97 95 95 17
IIID-23 100 100 100 100 - - - - - 100 10
D32 99 99 99 99 - - - - - 99 10
Notes: GRA I GRA-M-l
GRA II: GRA-M-3
GRA III: GRA-M 4
~*t~ indicates % cytotoxicity when complement
alone was used.
- 43 -
.... .. .. . . .
From the results shown in Table 15 a~ove it can be seen
that GRA-M-l, GRA-M-3 and GRA-M-4 lack H-2.
TEST EXAMPLE 9
C57BL/6 mouse was transplanted under S.C. with LLC (2x106)
from the same strain and after 6 days 1 ng (protein) of GRA-
.M-3 obtained in REFERENCE EXAMPLE 2-(d) was administered subcuta-
neously. Thereafter administration was repeated for 4 days
once a day at the same dosage. The day next the inal administ-
r~-tion t~m)or cells were excised and weighed. As a control
physiological saline administered animals were used. Each
test group comprised 5 animals.
As a result, average tumor weight of the control group
was found to be about 500 mg. In CRA-M-3 administered group
3 showed disappearance of tumor and two had average tumor
weight of about 100 mg.
TEST EXAMPLE 10
C57BL/6 mouse was immunized subcutaneously with 1 ng
(protein) of GRA-M-3 obtained in REFERENCE EXAMPLE 2(d) once a day
for 3 days and on day 5 spleen cells were collected from the
animal to obtain effector cells. A mixture of the effector
cells and Lewis lung carcinoma (LLC) as target cell in a ratio
of E/T = 50:1 was prepared and a portion (lx105) thereo~ was
*ransplanted to mouse of the same strain and Winn assay was
performed (J. Immunol. _, p,228-239 (1961)).
The results obtained are shown in Table 16 below.
Table 16
Day 20
Effector Tumor Growth Rate ~%)_ Mortality/
Cells Day 15Day 17 Day 18 Day 19Day 20 Group
A 5.75.5 5.7 3.1 1.4 0/10
B 21.439.5 37.1 55.4 76.8 4/10
C 39.365.1 61.7 9g.1 96,4 4/10
Notes: Group A represents spleen cells from GRA-M-3
immune mouse.
Group B represents spleen cells from normal
mouse.
Group C represen-ts spleen cells ~rom mouse
after 10 days from the transplantation of
LLC (lx106).
Tumor Growth Rate was calculated according to
the following equation:
Tumor Growth Rate ~%) = TA TN x 100
TN
wherein TA represents the thickness of the foot pad on the side
where the tumor was transplanted and TN represents the thick-
ness of the normal foot pad.
TEST EXAMPLE 11
C3H/He mouse was immuni~ed with 4.5 ~g (protein) of
GRA-M-4 obtained in REFERENCE EXAMPLE 2 ~d) and 0.1 m~:~of Freund's
Complete A~juvant at the sacrococcygeal portion. After two
weeks lymph node cells were collected by conventional me~hod
and were used as responder cell for determining prolierative
response by GRA-M-4.
For this purpose, responder cells (4x105) were incubated
in RPMI-1640 medium containing 15~ FCS in the presence of
GRA-M-4 for 5 days. During final 18 hours of the incubation
period 1 ~Ci of 3H-thymidine ~3H-TdR) was added to the medium
and its incorporation into the cells was counted.
The results obtained are shown in Table 17 below.
Table_17
Concentration 3H-TdR Incorporation
GRA-M-4 ~ng/mQ)~mean cpm ~ S.E.) S.I.
Control 05,302 _ 1,761
0.57,903 + 1,290 1.5
110~076 ~ 936 1.9
Experiment 5 10,686 ~ 429 2.0
2010,615 + 1,270 2.0
408,565 ~ 1,419 1.6
Note: S.I. indicates stimulation index in terms of
Experiment/Control.
TEST EXAMPLE 12
Delayed type hypersensitivity ~DTH) response of C3H/He
normal mouse, X5563 lmmune mouse and MH134 immwne mouse
obtained in the same manner as in EXAMPLE 5, and GRA-M-4
immune mouse and GRA-M-5 immune mouse obtained ln the same
manner as in TEST EXAMPLE 11 was determined by foot pad
reaction (FPRj. That is9 GRA or MMC-treated tumor cells
were challenged at the foot pad skin of the hind leg of the
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~o~
animal and swelling of the foot pad 24 hours after the
chalienge was determined. Degree of DTH response was calcu-
lated by subtracting the swelling before challenge from that
after challenge (lO 2 mm).
The results obtained are shown in Tables l8-22.
Table 18
Mean Foot Pad Increment (10_ mm)
Challenge Normal MouseMH134 Immune Mouse
_ . _ . _ _ .. ..
l 2.8 13.6
2 12.4 32.0
3 3.6 3.6
4 3.2 22.0
~ 6.8 27.6
Notes: Group l; syngeneic normal spleen lxlO6/20 ~Q
medium (Hanks solution)
l~ Group 2; MHl34 cell lxlO6/20 ~Q medium
Group 3; medium 20 ~Q
Group 4; GRA-M-4, 0.8 ~g ~protein)/20 ~Q medium
Group ~., " 0.4 ~g "
Table l9
Mean Foot Pad Increment ~lO 2 mm)
Challenge Normal Mouse X5563 Immune Mouse MHl34 Immune Mouse
l ~.4 4.3 l.l
2 0.9 - 24.3
3 2.0 16.9
Notes: ~oup l;~medium (Hanks solution) 20 ~Q
~ up 2; ~RA-M-4~ 4 ~g (protein)/20 ~Q medium
Group 3; GRA M-5,
38
Table 20
Mean Foot Pad Increment (10 2 mm)
ChallengeNo~rmaI Mouse GRA-M-4, Immune Mouse
1 1.7 -0.3
2 5.1 24.6
Notes: Group l; medium (Hanks solution) 20 LQ
Group 2; GRA-M-4, 4 ~g (protein)/20 ~Q medium
Table 21
Mean Foot Pad Increment (10 2 mm)
ChallengeNormal Mouse GRA-M-4, Immune Mouse
1-1.8 0.6
26.3 20.0
36.8 6.3
Notes: Group l; medium ~Hanks solution) 20 ~Q
Group 2, GRA-M-4, 4 ~g (protein)/20 ~Q medium
Group 3, 4 ~g (protein)/20 ~Q medium of fraction
which passed through PNA-column at the
time of GRA-M-4 (hereinafter "C.P.")
Table 22
Mean Foot Pad Increment (10 2 mm)
Challenge Normal_MouseGRA-M-4, Immune Mouse
1 2.4 -1.0
~0 2 2.6 17.7
3 2.4 1.8
4 S.5 18.9
- 48 -
Notes: Group l; medium ~Hanks solution) 20 ~Q
Group 2; GRA-M-4, 3.8 ~g ~protein)/20 ~Q medium
Group 3; 3.8 ~g (protein)/20 ~Q medium of "C.P."
above
Group4; 3.8 ~ (protein) of GRA-M-4 and 3.8 ~g
(protein)/20 ~Q medium of "C.P."
REFERENCE TEST
X5563 Immune mouse was obtained in the same ~anner as in
EXAMPLE 5. Spleen cells from ~his immune mouse were used as
effector cell and the effector cells (107) and target cells
(X5563, 105~ were together transplanted to mouse of the same
strain. Winn assay was performed in the same manner as in
TEST EXA~PLE 10.
The results obtained are shown in Fig. 23, in which the
- 15 abscissa indicates days and the coordinate shows mean tumor
size (cmZ) ~ S.E. and various marks represent the following.
3 ~ O: Group in which effector cells were not added~
o -~o: Group in which non treated effector cells were
~added.
~ : Group in which effector cells treated with
rabbit complement were added.
x x: Group in which effector cells treated with
anti-Thy 1 (New England Nuclear Co.,U.S.A.)
and rabbit complement were added.
0 ~ - : Group in which effector cells treated with
anti-Lyt 1 (New England Nuclear Co.~ U.S.A)
J~2~9~3~
and rabbit complement were added.
~: Group in which ef:fector cells treated with
anti-Ly-t 2 (New England Nuclear Co.g U.S.A.)
and rabbit complement were added.
From the results shown in Fig. 23 it can be seen
that Lyt 1 type T-cells play an important role in the mecha-
nism of _ vivo effector in tumor immunity.
Further, it is known that DTH response is mediated
by Lyt-l T-cells~ (J. Exp. Med, 1~3 p,l534-39 (1976)).
While the in~ention has been described in detail
and with reference to specific embodiment thereof, it will
be apparent to one skilled in the art that various changes
and modifications can be made therein without departing from
the spirit and scope thereof.
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