Note: Descriptions are shown in the official language in which they were submitted.
FIELD OF THE INVFNTION
The pxesent inven-tion relates to cancer cell-
combatting lymphocytes (hereinafter referred to as
"killer cells") and a process for the production thereof.
More particularly it is concerned with killer cells which
act specifically on cancer cells ha~iny a glycorelated
antigen derived from the cancer cells (this antigen is
hereinafter referred,to as "GRA"~ and destroy the cancer
cells, and a process for the production of said killer
cells. Addltionally the presen-t invention relates to a
novel anticancer agent, more specifically containing the
killer cell or GRA as an ac-tive ingredient.
BACKGROUND OF TIIE INVENTION
It is known that immune response effector cells,
particularly T lymphocytes playing a main role in cell-
mediated immUne response, cause rejection of grafts due to
foreign cell antigens, but exhibit no appreciable or very
limited immuno-inhibition against cancer cells. Thus, the
cancer cells are not destroyed and multiply in vivo,
finally putting the cancer-bearing host to death~
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1 However, the mechanism responsible to the
recognition of self from not seIf is not completely
clear and various investigations have been made to have
insight into the nature o~ material basis involved in
such system. For example, cell surface markers on mouse
leukemia cells or embryonal carcinoma cells using lectins
such as Dolichos biflorus agglutinin (DBA) and peanut
agglutinin (PNA) as described in'~l'o'c'h'e~'. Biop~y's
Res. Comm. 89 ~2) 448-455 (1979), Ibid.'96 (4) 1547-1553'
(1980), J. Biochem. 89 473-481 ~1981) and''Ce'11'18 183-191
September 1979.
~ However, as far as is known, there have been no
appreciable attempts to provide new lymphocytes that can
combat cancer ceils specifically and also provide anti-
cancer agents utilizing immune response of lymphocytes.
SUl!1r~ARY OF_ T~IE 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 the host and have very high immunogencity
~,'`!
1 that cause an immune response speciEic to the cancer
cells. Furthermore, it has been found that when GRA
is used to sensitize lymphocytes, there can be obtained
killer cells which act specifically on ~ancer cells
containing GRA and if the killer cells are administered
to the host, they recognize GRA and act on the cancer
cells containing GRA, destroying them, and thus that they
exhibit an excellent effect in the treatmen-t and preven~
tion of cancer.
` Therefore, the present invention provides
killer cells in one embodiment thereof.
~ In another embodiment, the present invention
provides 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. 1 is a microphotograph of ~audi cancer
cells;
Fig. 2 is a microphotograph illustrating the
formation of plaques by GRA-l-K-T of the cancer cell of
Fig. l;
Fig. 3 is a microphotograph of KATO-III cancer
~;!
cells;
Fig. 4 is a microphotograph illustrating the -forma-
tion of plaques by GXA-l-K-'I' of the cancer cell o~ Fig. 3;
Fig. 5 is a microphotograph of BT-I cancer cells;
Fig. 6 is a microphotograph showing the formation
~f plaques by G~A-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 o~ MOLT cancer cells;
Fig. 10 is a microphotograph sho~ing the formation
of plaques by GR~-l-K-T of the cancer cell of Fig. 9g
Fig. 11 is a microphotograph of BT-l treated with
a mi~ture of unsensitized human peripheral blood lymphocytes;
1~ Figs. lZ and 13 are each a microphotograph of the
cancer cell tissue o cancer-bearing mouce which is adminis-
tere~ ~ith GRA-~I-l-K;
~ig, 1~ is a miclophotograph showing the state o
cancer in a cancer-bearing mouse group which is administered
wi~h Gl~
h g. 15 is a microphotograph showing the state o:F
cancer in a cancer bearing mouse group which is not administer-
ed wi~h GRA-~
; . .
Fi~. 16 is a microphotograph showing the cancer cell
tissue of a cancer-bearing mouse group which is not administer-
ed with the GRA-~I-l;
Fig. 17 is a microphotograph of the cancer cell
tissue of the group which is administered with GRA-M-l;
~ ig. 18 shows ~n SDS gel electrophoresis diagram of
GRA using protein staining by C.B.B. me~hod;
Figs. 19 to 21 show S~S gel electrophoresis diagrams
o-E G~ using su~ar coloratlon by ~ASrr,etiiod;
1~ ~ig. 22 is a schematical illustration of the results of SDS gel
electrophoresis diagram o-f GRA using protein staining by C.B.B. method; and
Fig. 23 represellts a graph sho~ing tumor growth rate
in C31-l/}1~ mouse transplanted with X5563 immune mouse spleen
cells and X5563 cells.
DE'I'~_I.. ED DESCRIP' O_F r~lE INVEN'I'ION
l'he killer cells of the invention can be prepared,
for e,Yalnple, hy a method in which GRA is used to sensitize
lymphocytes.
GRA as used in the above method can be obtained
from cancer cells conta~ning GRA of humans or animals, e.g.,
cultivated cancer cells, transplanted cancer cells) SpOII-
-talleous]y-occuIring cancer cells, chemical substance or
virus-indllced cancer cells, and cancer cells derived from
operated tissues, by the rollowlng pTocedure.
.. . . . . . . . , .. .. . .. ...... ...... ... . . _ .. . .. . .. . . , . _ ..
~:
Cell membrane components are separated from cancer
cells as described above and treate~ with a lectin which
combines specifically with a terminal galactose or a terminal
N-acetylgalclctosamine, whereby GRA is combined with the lectin and
can be easily separated.
Suita~)le e~amples of the galactose-binding lec~in
include peanut lectin~ Ricinus communis lectin and Soybean
(Glyc:ine max) lectin (see J.B.C., 250p ~518-8523 ~1975~;
Bioche3TI. ~iophys. Res. Comm., 62, 144 (1975); Z. Immunitaets-
orch, 138, ~2~-433 (196g); Br. J. Exp _ hol, 279 228-236
(19~6); Proc. Nath. Acad. Sci. US~, 75, No. 5, 2215-2219 (1978);
Bioc.}lelllistry, 13, 196-204 (1974); and Carbohydrate Research,
51, 1~)7-ll8 (1976)). Suitable e~amples of the N-acetyl-
galactosalllirle-binding lectin include Dolichos bean (Dolichos
b:i1~us) agglutinin, braid orange lectin, Helix pomatia
lectin~ l:ima-bean (Phaseolus limensis) lectin, soybean
(Glycine max) lectin and Bauhinia bean ~Bauhinia purpurea)
lect:i.n.
The separation of the cancer cell membrane compo-
20 n~lltC; Call be achieved by known techniques such as a homogeni-
zation me~hvd and a solubilization method using a dissolving
agent. It is more advantageous to employ a method in which
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26~
1 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 dissolv-
ed in physiological saline or a buffer by the use of a
dissolving agent, and the supernatant portion is separated
by a technique such as centrifugal separation. Dissolving
agents which can be used include surface ac-tive agen-ts
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.), 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 combininy 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 lec-tin, an immune precipitation method using GRA
antibody or the like, a dialysis method, a gel filtration
method, an electrophoresis method, a physical precipitation
method using a sugar protein-precipitating agent, e.g., poly-
ethylene glycol and acetone, and a cornbination thereof.
More preferred is affinity chromatoyraphy utilizing a column
carrier containing lectin, and the column carrier can be
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easily prepared by immobili~ation of lectin on an insoluble
support. Such immobilization oE lectin on an insoluble
support can be performed by known techniques which are con-
ventiollally employed In immobilization o~ biosubstances.
OE ~hese techniques, i~ is preferred ~o use a cyan bromide
activation polysaccharide method and an immobilization method
using N-hydroxysuccimide esters. The cyan bromide activation
polysaccharide met}lod is a method in which an insoluble
su~port is`trea~ed with cyan bromide, and the activated
product thus obtained is subjected to a ~.oupling reaction
with lectin under rnild conditions to immobilize lectin~ In
treatinlJ the insoluble support with cyan bromide, -~or example,
basic compounds such as sodium hydroxide and sodium hydrogen-
carbonate are used to adjust the pH to rom 7~5 to 12, and
t}le support ;s treated in a sol~ent such as water, aceto-
nitr:i]e, or a buffer maintailled at pH 7.5 to 12, for example,
a U.l ~I sodium hydrogencarbonate buer (pH about 8.7) and
O.Ol ~l phosphate buf~er (pH about 7.7), at room temperature
or about l to 12 minutes. Usually the amount o the cyan
2~ bromi~le used is preferred to be equal to that o the insoluble
5-lpport.
~ny kno~n insoluble support ~Yhich is of lol~t non-
specific adsorption ~o all biosubstances, has high porosity,
; coatains a functional group capable of immobilizing bio-
~ 25 substances under mild conditions, and is che-nically and
- S
1 physically sufficiently stable can be used in the inven-tion.
Insolubl~ supports which can be used include ~ suppor-t made
of cellulose, e.g., aminoethyl cellulose, carboxymethyl
cellulose, bromoacetyl cellulose, and p-anilino cellulose,
a support of cross-linked dextran, e.g., SEP~IADEX* and CM-
SEPHADEX* (produced by Farmacia Corp.), and a support of
agarose, e.g., SEPHA~OSE* 2B, SEPHAROSE* 4B, and SEPHAROSE*
6B (produced by Farmacia Corp.).
In the couplin~ 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 thay are reacted in an appropriate
solvent such as a 0.1 mol/Q aqueous solution of sodiurn
hydrogencarbonate (containing 0.5 mol/Q of sodium chloride;
pH: 8.4) at a te~nperature of from 0 to 40C, preEerably
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 is trapped in the
column. Subsequently, substances capable of co~bining with e.g.
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~ S 2 ~ i
lectin can be passed through the column to perform an exchange
reaction, or alterna-tively an adsorption separator ~eluting
solut.iorl), e.g., a high concentration salt, an aqueous solu-
tiOIl of potassiwn thiocyanate, and a nitrate buffer, is passed
thrvug}l the column to`dissociate and obtain the desired GRA.
In the exchange reaction, examples of the subs~ances
capable of combining ~ith lec~in when a carrier for affinity
chromatograp}ly containing galactose-binding lectin is used
inclllde those ~hich 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 substanc.es capable of
combinirlg with lectin when a carrier for affinity chromato-
graI)}Iy containing N-acetylgalactosamine-binding lectin is
used incll~cle those which can combine with a terminal N-acetyl-
galactosamirle-coJ1lbining lectin, e.g., N-acetylgalactosamine~
disacc}lclrides containing a termirlal N-acetylgalactosamine and
oligvsacchtlrides containing a terminal N-acetylgalactosamine.
The thus-obtained GRA contains glycoprotein contain-
in~ a galactose and/or N-acetylgalactvsamine terminus, glyco-
li~)i~l aJId/or saccharide.
,
,
,~ , .
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.
~s~
1 The GRA thus prepared can be lyophilized, if
desired and further purified using ordinary separation
techniques. For example, those GR~ 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 ~y a separation method
using a galactose-binding lectin.
Lymphocytes as used herein are not critical,
and any lymphocytes of normal or cancer-bearing human
heing or animals can be used. Examples include those
- lymphocytes derived from perlpheral blood, bone marrow,
lymph node, spleenj 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
performed by cultivating the lymphocytes on a culture
medium containing GRA for a period of from 1 to 10 days,
preferahly from 2 to 7 days~
As culture media for use in the sensitization
of lymphocytes, there can be used various common nutxient
media which are conventionally used in such cell cultiva-
tion. Preferred examples include a RPMI 1640 medium, an
Eayle ~M culture, etc. with human serum, calf fetus serum
(FC5), calf serum, horse serum or the like added thereto.
The amount of GRA being added to the culture is usually
from 1 to 1,000
-- 1 1 --
I'
.
%~
1 ng/ml and preferably Erom 1 to 500 ng/ml, as calculated as
an amoun-t 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-KT*,
which is one of the killer cells of the invention, has
properties common to human peripheral blood T cells and
shows a cell-combatting activity which is specific to
cancer cells containiny GRA, which is shown in the example
as described hereinafter.
Typical examples of the novel killer cells of
the invention are GRA-l-KT and GRA-M-l which are prepared
in the examples as described hereinafter and all the killer
cells are available from the applicant.
The killer cells of the invention can be multi-
plied unlimitedly on the above-described culture medium
containing a T cell growth factor (TCGF*, lL-2). In this
case, selective cultivation of cloning of the killer cell
may be performed by the conventional ultra-dilution method.
These killer cells can be stored stably over a long period
of -time, for example, in :Li~uid nitrogen.
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1 The GR~ can be used singly as an active
ingredient and additionally, can be used in combina-tion
with other antibacterial ayents and cance~-inhibiting
agents. Cancer-inhibiting agents containing the G~A
of the invention as an active ingredient may ke in any
form as long as they are in the condition that the GRA
is contained in effective amounrts. 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, preser-
vatives, e.g., methyl~p-hydroxy benzoate, a stabilizer
which does not exert as such adverse influences on the
immunizing function of humans or animals, a buffer, and
the like.
Aqueous carriers which can be used include
physiological saline, and non-a~ueous carriers which can
be used include vege-table oil, e.g., sesame oil, mineral
oil, e.g., paraffin, animal oil, e.g., squallene, and
propylene glycol. In addition, for the purpose of `
immunological enhancement,
-e~
~ 5 ~ ~
appropriate adjuvants may be incorporated. Adjuvants ~hich
can be used include Freund's complete adjuvants, saponin for
anilnals, and aluminum hydroxide for humans.
The anticancer agent of the invention can be
administered on~e or repeatedly over a long period of time
to a cancer patient :Eor the ~reatment of cancer, or can be
admin;s-tered to one who is liable to suffer from cancer for
the prevention of cancer.
Since LD50 (mouce, intraperitoneal) of GRA is at
least 500 mg/kg as calculated as an amount o sugar, the
anticancer agent o-f the invention is of low toxicity and,
thereEore, it can be admillistered within a wide range of
dosage. Althollgh the concelltration of GR~ in the anticancer
agent of t}lf' invention is no~ critical, it is usually pre-
lS ferred to be rom 0.001 to 100 ~Ig/ml as calculated as an
amourl(: of sugar. ~ith regard to the dose o the anticancer
agellt, it is usually preferred that the agent is administered
in all a~)~ollnt of 0.001 to 1,000 ~Ig/kg/day, at the same time
or in seve~ral portions, although it varies depending on the
e~tellt of disease,and the age and sex of the patient.lhe thus-pre~arfd an~icancer agcnt containing the
ki.ller cell'as an active ingredient is preferably used as
an injectable solution in co]nbination with carriers which
are used in the preparation of such blood medicines.
Carriers as used herein are not critical, but carriers
.. - ... .. . .... ..
'''
6~
1 having a tonicity equal to that of blood are pref~rred.
II1 particular, physiological saline is preferred. In the
preparation of the agent, it is preferred that the killer
cell is sufficiently washed wi~h physiological saline or
the like to remove the above-described culture medium and,
thereafter, it is floa-ted in a carrier.
The concentration of the killer cell in the
agent is not limited specifically, but it is preferably
from 105 to 109 per milliliter. When the killer cell is
administered intraperitoneally in a dose of 108 per mouce,
no toxity is observed. Although the dose of the anti-
cancer 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 10 /kg/day, in one portion or in divided
portions.
The following example, reference examples,
experiment examples, and comparative examples are given
. to illustrate the invention in greater detail although the
invention is not limited thereto.
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 of a 0.01 M phosphate
buffer containing 0.85g~ NaCL (pH=7.2). In 1 ml of a
0.5 M hydrogen-
,~ .
526~
1 carbona-te 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 mlxture was then stirred at room -temperature
for 2 hours and, thereaEter, it was separated on SEPHADEX*
G25 (10 mm x 300 mm, produced by Farmacia Corp.). The
initial peak was collec-ted. FITC*/PNA ratio=l.0 (l)-B
Preparation of FITC*-Labelled Lectin (DBA-FITC).
In the same manner as in (l)-A above DBA-FITC*
was obtained usin~ DBA produced by E~ 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 (p~=7.2) by a centrifugal procedure
and then 100 ~1 of PNA-FITC*, DBA-FITC* or SBA-FITC*
(200 ~g/ml) as prepared in (1) above was added thereto.
The resulting mixture was allowed to stand at room temper-
ature 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%
NaC1 (pH=7.2) and, thereafter, the cells were placed on a
~lass plate and e~amined under a fluorescent microscope.
Mouse X5563 and mouse MH13~ 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
use~ are al.l known and have been o~tained from Fi~st Pat}lology
Laboratories, Medical De~ar~ment of IYiIgata University.
r Table 1
, . ..
- Positive Ratio of
Cancer Cells GRA (~)
-
PNA DBA SBA
Raji tBurl~itt lymphoma) 98.3 1.4
~auj i (Burkitt lymphoma) 93.1 5.2
BT-l ~Burkitt lymphoma) 50.1 0
P-12 (T-cell lymphorna) 44.3 6.7
~IOLT (I-cell leukemicL) 0.6 4.8
; Fujima~i tB cell lymphoma) 19.1 5.3
Oda tIg]) myelorna) 0.6 10.0
QG-56 (lung cancer, squamous) 70.4 2.0
PC-l tlung cancer, squamous) 78.4 0.
PC-:5 (lung cancer, adeI-locarcinoma) 77.1 0
QG-90 (lung cancer, sma].l cell) 68.0 0
PC-13 (1Ung cancer, large cell) 17.0 0
~1~-2 tskomach cancer, poor
d;.:f~ererItiatiorl) 63.7 0.1
K,~lO-LII tstoJnach cancer, signek
ring cancer) 57.3 o
~I~N-45 ~stomach cancer, poor
di~ferentiation) 1.0 40 3
~I~N-l tstomach cancer,
adenocarcinoma-squaiTIous~ 4.6 0 4
- 17 -
~ 5;2~
Positive Ratio of
Cancer Cells GRA (%~
.
. PNA DBA SBA
hlKN-28 (stomach cancer) 0.~ 0,1
M~ 7L~ ~stomach cancer) 0.5 0
MGII-Ul (urinary bladder cancer) 37,4
KU-2 ~urinary bladder cancer) . 4.5 21.4
T-24 turinary bladder cancer) 14.6 0
NBT-2 (urinary bla~lcler canc.er)l3.l l.
NRC-12 (renal cancer) 23.9 0
~U-l (renal cancer) 3.3 0.6
Kuraml)cIli (ova~ian cancer) 80.0
i~ 1 (neuroblastoma) 50.9 1.7
YT-nll (neuroblastoma) 3.6 0.5
TGW--~Iu~ TI eul~oblastoma) 4.l 0
TG~-r~u-ll ~neuroblastoma) 2.0 l.n
GOTO (neuroblastoma) 0.5 0
ITO (embryonal carcinoma) 96.9 12.3
NEC 8 (embryonal carcinom~) ~4.6 0
SC~I ~choriocarcinoma7 stomach)1~.6 3.1
GCII ~chriocarcinoma, uterus) 5.4 0
Yl~-1. t1'habdomyosarcoma) 5.7 1.7
~Iousc X5563 (plasmacytoma) 92.0 0 90.6
Mouse ~I134 (hepatoma) 18.6 0 6.4
- 18 -
. . . . . ..... .. . .. . . . , . . . .. . . .. , .. . ~ .. ... .. . .. .. . .. . .. .
-
(b) The malignant tissues fro]n cancer patients were
passed t]lrou~ll a stainless steel mesh ~150) to make slngle
cell sus~ension. ~ftéT ~ashing twice with 0.01M 'I`ris-HCl
buffcr ~pll=7.4) containing 2mM CaC12, 2m?l~lgC12 and 0.85~
s N~Cl, 5x105 cells were resuspended in 100 ~1 of the buffer.
One hllndred ~1 of FITC-PNA or ~ITC-DBA (200 ~g/ml) was added
~o the cell suspension and the mi~ture was incubated at room
temperature for 20 minutes. A-lter washing three times with
cold P~;S, *he cell were observed under a -fluorescene micro-
scope.
The results are shown in Table 2. The malignant
tissues ~rom cancer patients have been obtained ~rom Kansai
Medical University.
Tclble 2
.
GRA
~o. Cancer Patient Tissue P~.~ DBA
1 Stoln.-ch cancer + -~
2 Stomach canccr + +
3 S-tomach cancer - -
4 Stomach cancer +
Stomach cancer + -~
6 Stomach cancer +
7 Breast cancer -~ -
8 Breast cancer -~ -
9 Breast cancer +
Breast cancer
- 19 -
. .. . .... . ~ .. ... ... . .. .... .... - . .
26~
GR~
No. Cancer Patient Ti.ss~Ie PNA '~ DB~
11 Colon cancer +
12 Colon cancer -
13 Esop~Iagus cancer
14 Hepatoma
Note: Symbol "~" indicates GRA is expressed on the
cell surface.
Symbol ~'-" indicates GRA is not expressed on the
cell surface.
REFERENCE EXA~IPLF. Z
Prepalcltioll of GR~
. . ~
~1) A Preparc-tion of In~obilized Lectin (PNA-Sepharose)
ThreP grams o~ CNBr-activated Sepharose ~B (pro-,
duced by ~:a~macia Corp.) ~as fully ~ashed ~ith 1 mM HC1 and
suspeIlded in 200 ml of 0.1 hI sodium hydrogencarbonate ~p~I=
8.5). l'hcn, 5 ml of a 0.01 ~I phosphate buffer ~pI-I=8.5)
corItaillin~. 20 mg of PNA was added, and they were reacted at
25"C I.'or 2 hours while sometimes stirring to prepare PNA-
Sepharo s e .
(:l)-B 'fn the same manner as in (1)-~ above except that DBA
is used ir~s~ead' of PNA, DBA-Sepharose l~as obtained.
~2) PTeparation of GRA
(a) BT-l (Burk;tt lymphoma) cells (1.3x108) were washed
th~ee times wi~h physiological saline, and 30 ml of a 0.01 M
tris hydrocIlloric acid buffer (p~-I=7.~) containing 290 oE
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, . . . .. . . . . .
~gs~6~
"TR~10N X-l00" (produced by 1Yako Pure Chemical Industries
I.td.) 3 O. 85% of NaCl, 2 m~l of CaCl29 and 2 mM of ~1gCl2 was
adcled thcreto. The mixture was stirred at 4C for 15 minutes
and the~ as s~1bjected to ultracentrifugal separation at a
S rate of l00,000 x g. Of 28 ml of the thus-obtained super-
natant liquid, a l4 ml portion was passed through a column
~diameter 0.5, length l cm~ for a-ffinity chrornatography,
packed with PNA-a~arose beads (produced by Maruzen Co., Ltd.~
which had been equilibra-ted by a tris hydrochloric acid bu-Efer
(p~1=7.4) containing 0.1% TRITON X-l00, 0.85~ NaCl, 2 m~l CaCl2,
and 2 nu~l MgC]2. A~ter being washed Wit}l the same buffer as
used above, ic ~as eluted with a 0.0l M tris hydrochloric
; acicl bl1f:Ee,f (p~1-7.4) containing 0.l M lactose, 0.85~ NaCl,
2 MM CaC12, 2 n~ i~1gCl2, and 0.1% TRITON X-L00. The thus-
lS eluted portion ~as dialyzed with a 0.0l ~1 tris hydrochloric
acid b11ffer containing 0.85% NaCl, 2 m~1 MgCl2, and 2 mM CaCl2
for ~8 hol1rs to obtain 17 ml of a GRA solution. With the G1
solutior1, the a111ou1lt of protein and the amount of sugar were
Tneasure~d hy the ~olin-Iowry met}lod and the phenol-sulfuric
acid n1ethod, rcspectively, and they were found to be 6~4 ~g
a1ld l20 ~1g, respective:Ly. This is hereina~ter referred to
a~i "GR~-:I".
(b) C311/1-1c ~1ouse 1nam111ary tumor tM~1T) cells tlxl0l)
were washed three times with physiological saline, and 30 ml
of a 0.0l ~ tris hydrochloric acid bufer (pll=7.4) containing
- 21 -
2Qo TRIlON X-100, 0.85% NaCl, 2 m~l CaC12, and 2 m~ ~IgC12 was
added thereto. The mixt~lre was stirred at 4C for 30 minut~s.
Subseq~lently the mixture was subjected to ultracentrifugal
separation at a rate of 100,000 x g for 2 hours, and the
supe~natant li.quid was dialyzed ove~-night with a 0.1 M tris
hydroclllor]c acid buffe~ ~pH=7.4) containing 0.85% NaCl~ 2
mM CaC12, and 2 mM MgC12. The thus-di.alyzed liquid was
- conentrated to 3 ml, and a 1 ml portion was then passed
throug}l a colu~ln (diarne-ter 0.5 cm~ length 2 cm) for affini~y
chromatograp}ly, packed with the same ~'NA-Sepharose as used
above which had been equilibrated with a tris hydrochloric
aci.d b~.~fer (pH=7.4) containing 0.005% TRITON X 100~ 0.85%
~laCl, 2 mM CaC].2, and 2 mM MgC12. After being ully washed
with the saJne buffer as used above, it was eluted with a
lS 0.01. ~I tris hydrochloric acid bu-ffer (p~-1=7.4) containing
0.~ aCtOSe~ 0.85% NCLC1, 2 m~l CaC12, Z n~M MgC12, and 0.005%
TRITO;~ X-100, and -the thus-eluted portion was dialy~ed for
4~ hol.~rs W:it]l a 0.01 ~I tris hydrochloric acid buffer (pH=7.4)
contcl:irling 0.85% NaCl~ 2 mhl CaC12 and 2 n~l MgC12 to obtain
2 ml oE a GR~ so].ution. With the GR~ solution thus obtained,
the alnoullt of protein and the amount of sugar were 156 ~g
and ~ g, respecti~ely. This i.s hereinafter referred to
as "GRA-M-lt'.
,
,, .
- 2Z -
, .
~ 5 ~ 9
(c) ~ppro~imately 120 g (wet ~eight) of KATO-III was
a homogenized in 1~0 mi o PBS by a ~aring blender. After
. centTi:fugation at 100~000 g for 1 hour, the pellet was dis-
solved ~ith a i.n 100 ml o.E 2% TRITON X 100 in 0.01 M Tris
HCl buffer ~pH--7.6) con-taining 0.15 M NaCl.. The supernatallt
collec~ed by centrifu$ation at 100,000 g fQr 1 hour and
applied to a column of PNA-Sepharose 4B (0.8x15 cm) equili-
brated ~ith 0.015% TRITON X-100 in 0.01 M Tris HCl (pH=7.6)
cont2irliJIg 0.15 M NaCl. After wash:ing with 50 ml oE the
buffer, the GRA were eluted with the buffer containing 0.1
M lactose. The eluted GR~ dialysed a.gainst 0.85~ NaCl,
concentrated by Sephadex Pharmacia Co. and stocked at -20C
before use. ..
The amourlts o:E protein and sugar determined in the
].5 same manner as in (a) above were ~ound to be 2.0 mg and 0.8
mg, respective.l.y. Ihis is hcreinafter refcrred to as "GRA-Z"
(d) In the same manner as in (c) above, thc follo~;ng
GRA samp].~s were obtairled.
~3 -
~95~
Table 3
GRA
GRAMaterial Protein Sugar
Sample SourceAmount Conten~ Content
~g) (mg) (mg)
GRA-3 Bl-l 33 0.5 0.09
GRA-~ Breast Ca. 5 0.24 0.5
(excised)
GRA--5 QG-56 24 0.6 0.38
GRA-6 QG-90 26 1.0 Q.54
GRA-7 Raji 29 0~78 0.45
GRA M-2 hlMT 200 11~3 Z8.4
GRA-~l-3 LI.C 14.4 0.06 0.07
lU GR~-~l-4 MH-134 85 0.65 0.35
GRA-~1-5 X-55G3 25 0.56 0.23
(e) In the same manner as in (c) above except that ~IKN-~5
(about ~9 g) was used instead of Kato-III and DBA-Sepharose
o~?tai.ne~l iJI (l)-B above was used instead of P~A-Sepharose 4B
; 15 and e:lution was carried out with N-acetylgalactosamine, a
GRA preparcltion havin~ a protein content of 0.03 mg and a
sugc!r con~eilt of 0.01 mg was obt~ined. This is hereinafter
rcferred to as "GRA-8".
(-f) GRA-3 prepared iII (d) above (5 mQ) was charged in DB~-
Se~)}laroselc,olumn and elu-ted with Tris-HCQ buffer (0.015%
Triton X-lO0, 2 n~l MgCQ2 CaCQ2, 0.85~ NaCQ) to obtain 4 mQ
frac~ions Nos. 1-12. Frac-tions Nos. 1-3 are termed "G M -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
- 2~ -
,, . .. . . , . .. ., ., , ,, . .. 1~ .~.. ..... ...... .. ....... ... ..... ..
.
to obtain fractions, which is termed "GRA-3-C".
~g) SDS Gel Electrophoresis
Each of the GRA preparations accordi~g to the above
procedures w~s subjected to electrophoresis according to the
met}locl described in ~airbanks et al: Bioc]-emistry vol. 10
p.2606, 1971.
'E`he results obtained are sho-~n in Figs. 18 to 22.
In Figs. 18-1~, numerals 1 to 5 indicate the following.
1 --- standard; 2 --- GRA-~l-3; 3 --- GRA-7;
4 --- G~-1; and 5 --- GRA-2
In Fig. 20 numerals 1 to 4 indicate the following.
1 --- standard; 2 --- Gr~ l-2; 3 --- GRA-6;
4 --- G~-5
In Fig. 21 Jlwnerals 1 to 3 indlcate the following.
1 --- GRA-M-4; 2 --- GRA-~1-5; 3 --- s~andard
In ~ig,. 22 n~ erals 1 to 4 indicate the Eo],lowing.
1 --- (~RA-3; 2 --- GRA-3-A; 3 --- GRA-3-B;
'1 --- cr~A~-3-c
Eig. 18 shows the state of GRA s~bjec~,ed,to
prot~irl stn~ ing reactioll according to C.B.B. method
(Fairba~ s et al: Blochem try vol. 10 p.2606, 1971).
E:igs. 19-21 shows the,state of GE~A subjected to
sugar color reaction according to PAS method (R.M. ~acharius
et al: Anal ~iochem. ~ol. 30 p.148 (1962)).
~ig. 22 shows scherna-tical illustration o-f the results
o staining according to ~l~e C.B.B. method described above.
- 25 -
$~
For the standards substances listed below from
Biorad Lab. Cali. U.S.A. were used.
200 K dalton; myosin
116 " ; ~-galactosidase
92.5 " ; phosphorylase
66.2 " ; BS!~
~I ; ovalbumin
2~.5 " ; soybean trypsin inhibitor
RF;F:E-RENCE EXQ~IPLE 3
Preparation of TCGF
~a) The spleen (4 ~g) of Japanese monkey (obtained rom
Japan Plymates Co., Ltd.) was excised and washed twice with
a I~P~ 1640 culture medium (produced by Flow Laboratory Co.,
L.tcl.). Cells were iltered by the use of ~5esh (produced by
Milipole Inc., 150 mesh) and were subjected to a specific
gravity centri~ugal method (specific gravity, 1.076) ~o
o~tai]-l 2 liters of 2xlO9/ml o~ lymphocytes. The thus-
obtaille~l lymp]locytes were ~ashed three times with a RP~II-1~40
CU].t~ll't' medillm~ and the lymphocyte number was adjusted ~o
SxlO per milliliter by the use of the same medium as used
al~ove, containin~ 10% FCS. It was -then allol~ed to stand in
a carbon dioxide ermentation apparatus at 37C for 1 hour.
- 26 -
. , . .. . , . . . ...... ., .. . . , .. .. . .. , . .. .. ... .. ,.. " ,. , . ,.. , . ~, .. , .. , .. .. ,, .. .. , .. _ .,
.' ' " ' '
~3~
The sup~rnatant lymphocytes ~ere recovered and the lymphocyte
nwllber was adj-lsted to lx106 per milliliter by the use of the
same cul-ture medium as used above 7 containing 1% FCS. Sub-
sequell-tly 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
fer~nentation apparatus at 37~C for 48 hours. A cen-trifugal
se~aration procedure (3,000 rpm) ~as applied for 10 minutes,
and -t}le resulting supern~tant liquid was recovered and
sterilyzed by filtering with a Millipore filter ~produced by
Millipore Inc., 0.2 ~m~ to obtairl 2 liters of TCGF.
, (b) The source of TCGF was the supernatant of mix-
culturecl peripheral blood lymphocytes of lO healthy donors.
Non-ad~lerellt lymphocytes prepared fTom C.F, (Conray. Ficol
(Japall Inununoresearch Co.)) separated lymphocytes by adsorp-
'tiO]I OJI plastic surface at 37C for 1 hour were suspended in
~PMI 16~0 medium contain;ng 1% FCS (1.5xlO~ cells/ml) and
incubat:ed with 0,2% Pll~-P~ indomethacin ~1 ~g/ml) and human
. r
B-ce:l:l line (BT-l) (1,5,~'l0'' cells/ml) pretreated w:;th mito-
~nycin C ~50 ~Ig/ml). ~fter ~8 hours, the culture supernatant
was harvested and used as TCGF source. ~I. Inoue et al~
Scancl J. Immunol. 12 p.1~9-15~ ~1980))
.
.
5~
1 REFERENCE EXAMPLE 4
Preparation of ~ymphocytes
(1) Human Peripheral Blood Lymphocytes
Blood t50 ml) obtained from a healthy adult or
various cancer patients by heparinization was subjected to
centrifugal separation by the use of FICOL PACK* ~produced
by Farmacia Japan Co~, Ltd.) to obtain 5x107 of peripheral
blood lymphocytes.
(2) Mouse Spleen Lymphocytes
The spleen of a C3H/He mouse (male, 6w) was ex-
cised and washed twice with a RPMI-1640 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 4x107 of spleen lymphocytes.
EXAMPLE 1
GRA-l (amount o 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 a
RPMI-1640 culture medium containing 15% FCS to prepare a
sensitization culture medium.
Human peripheral blood lymphocytes (5x106/5 ml?
as obtained in Reference Example 4 ((1)) was added to 5 ml
of the sensitization culture medium as preparecl above which
*Trade Mark
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1~"
i;26~
1 was placed on a laboratory dish, and cultivated at 37C
for 2 days. They were fur-ther cultivated on the RPMI-1640
culture medium containing 20% TCGF* and 15% FCS*, as ob-
tained 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 mouse spleen lymphocytes as obtained in
Reference 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 so that the final amounts of protein
and sugar were 1.5 ~g/ml and 0.9 ~g/ml, respectively. A 5
ml portion of the resulting mixture was cultiva-ted on a
laboratory dish (60 mm x 15 mm, produced by Falcon Co.)
at 37C for two days. The formation of cloning was observ-
ed. The portion was further cultiva~ed on a RPMI-1640
cul-ture medium having 15% FCS containing 20% by volume of
TCGF* (produced by Japan Immuno Research Laboratories
Co., Ltd.) Eor additional 4 days to obtain 50 ml of a
killer cell solution containing lx106 killer cells per
milliliter. This is hereinafter referred to as "GRA--M-1 K-T".
*Trade Mark
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2~9
EXA~IPLE 3
PeIipheral blood lymp]locytes ~5xl06) of healthy donor was
lncubated in RP~ 1640 medium containing S0 ng/mQ ~protein
COll~:'ilt) 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 20~ and incubation was continued for fur-
thcr 3 days to obtain killer cells. This is hereinafter
referred to as "GRA-2-K-l".
EXA~IPLE 4
~iller cell prepara-tions GRA-8-K-T, GR~-3-A-K-T and
GRA-3-C-K-T were prepared in the same manner as in EXA~IPLE 1
using GRA-8, GR~-3-A and GRA-3-C~ respectively ~protein
conterIt: 50 ng/mQ) and the TCGF obtained ;n REFERENCE EXA~IPLE
3-(b).
EXAMPLE 5
C:3II/~]e mice ~female, 8 weelcs) was implanted intradermally
wi.th XS563 cells ~l06) of the same strain and after 7
days the tumor was excised surgically. A-fter another 7 days
X55fi3 cells ~l05) oE the same strain was inoculated~
and mice resi.stant ~o inoctllation ~lere named immune mouse.
e spleen cells of the immtlne mice and C311/~ie normal
mice were prepared according to commonly used method.
Each lot of spleen cells ~5xl06twell) was sensiti~ed
, ~ Wit}l 40 ng/m~ (protein content) of GRA-~I-5 in RP~II-l640 medium
, ~ ~S containing 15% FCS for 5 days to obtain killer cells.
; Killer cell preparation obtained from normal spleen cells
,~ .
, - 30 -
., .
, . .
~, .
is hereinafter referred to "GRA~ 5-K-T-l" and th.-t obtained
from immune spleen cells is hereinater referred to "GRA-~1-5-
K-T-2".
EXA~IPLE 6
I-lurnan pcripheral blood lymphocytes ~5x106) weTe incubated
in 5 mQ of RP~ 16~0 medium containing 50 ng/mQ (protein
content) of GRA-l at 37~C ~or 2 days. On day 3, the lympho-
cytes were t~ansferTed to RPMI-1640 medium containing 10%
serum froln the donor of the lymphocytes and 0 to 100 ng/mQ
~protein content) of GRA-l and incubation was continued for
~urther 5 days to obtain killer cell preparations shown in
Table ~ belol~.
1'ab].e
GRr~ Concentration ~n~/mQ)
In:~t al Dly Day 3 Ki_ ler Cell
0 GRA-l-K-T-l
1.6 GRA-l-K-T-2
3 . 2 GRA-l-K-T-3
6 GR~-l-K-T-4
:l2 . 5 GRA-l-K- r-5
GR~\-l-K-T-6
GR,~-l-K-T-7
SU 100 GRA-l-K-T-8
_X~IPLE 7
; (~) Peripheral blood lym~)}locytes (PBL) frorn various cancer
patien-ts after operation ~re sensi-tized with GR~-3 to obtain
~s~
l killer cell preparations. PBL (5X10 ) from the patien-ts
were incubated in RPMI-1640 medium containing 50 n~/ml
(protein content) of GRA-3 for 2 days and then in RPMI-
1640 medium containing 20% TCGF and 15% FCS for further
5 days to obtain killer cells shown in Table 5
Table 5
Periphera'1 Blood Lymphocytes
, ~ays 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-X-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 operation) from the cancer patients from whom PBL was
collected as determined in the same manner as in REFERENCE
EXAMPLE l, 2-(b). P-GRA and D~GRA are results obtained
using FITC-PNA and FITC-DBA, respectively. The days after
operation indicate the timing when PBL was collected after
the operation.
(b) PBL (5X106) collected from breast cancer patients
~ 25 after 2J days from the operation was incubated in RPMI-
',, 1640 medium containing 50 ng/ml (protein content) of GRA-
, 3 and 10% serum from the patient for 7 days to sensitize
', PBL and obtain killer cell preparation. This is
hereinafter referred to "G~A-3,-K-T-7"
.~
- 32 -
~;!
':
-
EXA~IPLE 8
G~ K-T {lO8) as obtained in Examp]e l was dis-
solved in lO ml of physiological saline to prepare an injec~
tabl~ solution.
EX~1PLE ~
- ~i) GRA--~1-l as obtained in Refere-nce Example 2 ((2~-(b~)
~as dilu-ted wit11 p11ysiological saline so that the amounts of
sugar and protein be l 0 ~g/ml and l.6 ~g/ml, respec-tively,
to thereby prepare an anticancer agent No. l.
(ii) A tur1lor lump of C3~1/He spontaneously occurring breas-t
cancer was sterilely cu~ to 5 mm cube clump and transplanted
to un~ler the back skin of each o~ -ten C~S~ le mice of the same
strai1I as above (7-week-ave, males). Seven days after the
trans~ ntation, the fixation and multiplication of the tumor
were confir1Iled. To five o~ the mice were each administered
subcutaneously -t31e an-ticanct-~r No. l as prepared in (i) above
in a dosage o:E 300 ~l per day at two day inter~als. The
remailLilIg ~;ve 1IIice were usetl as untreated çontrols. Ten days
a~ter -tlIc first adminis-t:ration7 the tumor was excised by ope-
ration, an~ the ]nean Weig>}lt ~as measured. At the same -t;me,
pathohistological examirlation was performed.
Tumt) L- VO lume:
Admins-tered group 22.3 mm3 (Fig. l~)
Control 162.7 mm3 ~Fig. 15)
This means t]lat ~here was a 86.3% reduction in t31e-t~nOr
Pathohistological E~amination:
"
- 3~ -
%~
1 In -the con-trol group (Fig. 1~), birds' nest
bodies of cancer were formed, the type of tissue was like
medullary canalicular cancer and the multiplica-tion 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 fibro-
sis occurred, leaving only a very limited amount of cancer
cells. Thus, the anti--tumor properties of ~he anticancer
agent of the inven-tion were observed.
TEST EXAMP~E 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 a-t 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 buffer (pH=7.2) with 0.~5%
NaCl added were added, and the plate was subjected to a
centrifugal separation procedure at a rate of 600 rpm for
5 minutes. The plate was then reversed, and unreacted
SRBCN was removed. A dyein~ solution (Brilliant Cresyl
Blue, produced by Merck & Co.) was added to dye the ]ympho-
cytes, and rosette-forming positivity was examined. As
a result, it has been found that
*Trade Mark
- 3~ -
~4~l~
3%~
at least 98~ exhibits rosette-forming positivity (T-cells.)
TEST EX~1Pl.E 2
~ci-fic Cancer Cell-Killin~ Activity
(a) of the cells shown in Table l, the following five
cell strains having different GR,~ positi~ity ratios were
used as target human cancer cells.
Target Cancer Cells:
~o. l BT-l (Bur~itt lymphoma)
~Jo. 2 Daudi (Burkitt lymphoma)
No. 3 K~TO-III (stomach cancer)
No. 4 ~IKN-45 (stomach cancer)
No. 5 ~IOLT (T cell leukemia)
On a microplate (produced by Falcon Corp.~ ~ere
laminated 5xl04 per well of target cancer cells by a centri-
fugal procec1u~ at a rate o ~00 rpm for 5 minutes. Then,
4xl03 per wel] of ~T~-l-K-T as obtained in Example l was
gently added an(1 incubated for l hour.
The killing activity was determined according
to the d~gree of plaques-formation, and was rated as follo-rs:
~ . Killing activity is significantly observed.
ICilling activity is observed.
ICilling activity is slightly observed.
- Killing activity is not observed.
In a con~rol groul), there were used unsensitized
human peripheral blood lym1hocytes which had been prepared
- 35 -
', ,
i;26~ '
in t]le same manner as in Example 1 except that ~RA was not
used. The results are ShO~iJI in Table 6.
It is apparent from Table 6 that the killer T cells
produced by the process of ~he inv~ntion have strong GRA-
speci~ic cytotoxic activity.
I`able 6
Target Cancer GRA Positivity Determination of
Cell Ratio (%) Plaaue-~ormation
. _ . . . .. _
CR~ -T ~Daucli ~ig. 1) 93.1 ~ (Fig. 2)
Group
KAT0-III (Fig. 3) 57.3 ~ (Fig. ~)
BT-l (FIK. 5) 50.1 ~-~ (Fig. 63
~I~N-45 tFig. 7) 1.0 ~ (Fi~. 8)
~I~L'I' (Fig. 9) 0.6 - (Fig. 10~
Colltro:l BT-l 50.1 - (Pig. 11)
Group
: (b) Tlle salne -targe~ cancer cells as used in ~a) above
; 15 (3.2x106) were mixed with 8x105 GR.~-l-K-T (cell ratio: 5/1)and the resultin~ c~ll mixture (total numbe~: 4x106) was
culti.vated on n RYMI-16~0 med;urn contai.TIing 15% FCS. A~ter
one hour, th~ number of remaining cells was counted and the
% cy~otoxicity was cal.culated by the -following equation.
~ I number of cells ater
% cytotoxicity = (1 ~ (numbeivraofo-n~lls be~or~e3 x 100)
cultivation ~4x106)
The resul~s are shown in Table 7.
. ~ 36 -
Table 7
Numb~r of Cells
Belore Culti- i~fter Culti-
Tar~et Ce11 vation. vation % Cytotoxicity
Daudi4xl06 2.9~l06 2~
KATO-III~ 4~10 3.7xl06 7.5
BT~ o6 3.2xl06 20
~IKN-454xl~6 3.9xl06 2.5
~IOLT~l06 4.2xl0~ -5
(c) The procedure of (b) above was repeated with the
exception that the mi~ing ratio of GRA-l-K-T to target cancer
: l0 cell ~s changed to 5f3. T1le results are sho~n in Table 8.
Table 8
Numher of Cells
Before Culti- A~ter Culti-
Ta~et Cell vcLtion vation % Cytoto~icity
Daudi ~xlo6 3.6~105 91
KATO-III 4x]o6 3.6~106 24
BT-l 4~lo.6 l ~cl06 65
N-~5 ~xlo6 3.7xl06 7.2
~IOLT 4xlo6 4.lxl06 3
In ~hc~ above tcst it is obser~ed tha~ GRA-l-K-T shows a high
binding activit~ to Daudi, I~ATO-III and BT-l but only a low
bi.n~ing activity to ~IA~--45 and ~IOLT.
TESl` EX~~1Pl.E 3
C311/~le spontaneously-occurring breast cancer-bearing
~ice ~ere adn1inisterecl subcutaneously by GRL~-~1-l-K-T as obtained
- 37 -
.. . . . ... ......... _
2~
;n Example 2 at a dosa~e of ~x106/0.3 ml/mouse three times per
week every other day. After tcn days, the focus was taken out
and examin~d.
As shown in Fig. 12, the infiltration of lymphocytes
in-to cancer cells occùrred, and the breakage oE the tumor area
was observed. ,~lso, from Fig. 13, it has been observed that the
calc;.fication of the tumor area occurred~ and tnus it can beseen
that the killer cells of the invention have antitumor activity.
COMPAR~TIVE EXA~IPLE 1
In this example, cancer cells p se were used as specific
antigens in place o~ GT~ for use in th.e process of -the in~ention.
Cancer cell-scnsitized lymphocytes were obtained in
the same manner as in Example 1 except that, in place of ~RA,
BI'~ audi, ~'lO-III, or MKN-45 was used at a le~el of lx105 per laboratory di.sh.
ith these lymphocytes, the cytotoxic activity ~as
examined in the same manner as in Test Example 2 ~a)). The
resull;s are sho~n in Table 9.
It can be seen from Table ~ that the above-prepared
l~nphocytes clo not have any cytotoxic activity.
'lable 9
Cells for Use in Sensiti-
zation of Lymphocytes
TarPet Cell BT-l Daudi ~T~rIT~ s
BT-1
Daudi - - - -
~ATO-IIX
~lKN-45
- 38 -
~95:26~
TEST EXA~IPLE 4
In the same manner as in TEST E~`.IPLE 2-(b)~ the cancer cell-
killing activity of GRA-8-K-T, GR~L~-3-A-K-T and GRA-3-C-K-T
obtaine~t in E~A~IPLE ~ was determined. The results ob~ained
are shown in Table 10.
Table 10
Target ~ Cytotoxicit
Cell GRA-8-K-T GRA-3-A-K-T GRA-3-C-K T
K~TO-III ` 8.0 5.0 5O0
BT-l 4.3 5.0 4.0
~I~N-45 20 5.0 20
~iOLT 0 0 0
TEST EX~IPLE 5
(a) Cytotoxic;ty of each o~ killer cell preparations obtained
in EXAI'~PI.E 6 was determined by 51Cr release test (J, _m~unol.
122, 25%7-2533 (1979 ). That is, 50 ~Ci o~ radioac-tive 51Cr
~Japall Isotope ~ssociation~ was actded to KATO-III (2x107) and
the c~lls were incubated at 37C for 1 hour in RP~II-16~0
medium and suEiciently washed by centrifugation to ob~ain
51Cr-la~eled target cells. Killer cells (e~fector cells)
~2xl06) were adcled to the target cells (lx105) tthus E/T =
20/.l) ~nd ~he mixture was incubated at 37~C for 4 hours in
RP~II-1640 medium. Su~ernatant was collected by centrifuga-
tion anct its radioactivity was determined by liquid scintilla-
tion counter.
.
- 39 -
' , ' .
'
- The specific 51Cr Release (%) which corresponds to the
cytoly-tic activity of effector cells was calculated according
to the follo~ing equation.
S})ecific 5lCr Release ~%)
~ e]edse in I`est) - (S~ontaneous Release) x 100
(hla~imum Release) - (Spontaneous Release)
~The maximum release indicates the radioactivity when all the
cells are lysed).
The results obtained are sho~n in Table ll~
Table ll
Specific 5lCr
~iller Cell Release (%)
-
GRA-l-K-T-l 13
GRA-l-K T-2 25
GR~ -T-3 22
CRA l-K-T-4 20
GRA-l-X-T-5 22
(;RA-l-K-T-6 25
~RA-]-~-'r-7 27
C.T~-l-~-T-8 32
~rom tlle results shown iTI Table 11 above, it can be
s~en that T('GE: and FCS have no relation with the in~luction
of killer'cells.
, ~b) Cytotoxicity o~ G~-Z-K-T obtained in EXA~1PLE 3 was
', deteTmined by 51Cr release test in the same manner as in ~a)
above. Specific 5lCr relcase of 14.3% ~Yas observed on 51Cr-
labeled KATO-III as target cell ~E/T = 20/l).
- 40 -
., .
,
6~
TEST EX~MPLE 6
~a) Using ~ATO-III (E/T = 20jl) as target cell cytotoxicity
o kil:Ler cells obtained in EX~IPI,E 7-(a) was determined in
the same manner as in TES'r EX.~IPLE 2-(b).
Thc results obtained are shown in Table 12 ~elow, . rabl e 12
Killer Cell % Cytotoxicity
, _ _
GRA-3-~-T-1 38.0
GR~-3-K-T-2 18.2
GR~-3-K-'~'-3 20,9
GR~-3-~-T-~ 23,2
GR~-3 K-T-5 2~.5
GRA-3-K-T-6 20,2
(b) Cytotoxit,:ity o:~ GRA-3-K-T-7 obtained in EXA~IPLE 7-(b~
was deterJIlined usillg 51Cr-~ATO-III (E/T -- 20/1) as target
cell in -the same manner as in TEST EX.~MPLE 5. Specific 51Cr
release o the kil'ler cell was -Eound to be 25.5%.
TEST X/\~IPI,E 7
Cytoto~icity o~ the IciLler cells obtained in EXAMPLE 5
~as determined by 51Cr re:Lease test in the same manner as in
T~ST EX~IPLE 5. The target cell used was 51Cr-labeled X5563
cell. ~s a control lymphocytes obtai.ned in the same manner
as in EX~MPLE 5 except that sensit.ization of spleen cells~as carried
out Wit]l lxlO5/well o-f mitomyci.n C-treated X5563 cells (5xlO6/m~
of X5563 cells were trea-ted with 50 ~g/mQ of Mitomycin C Eor
60 min~l-tes) inste~.d of G~ M-5 were used,
- ~1 -
~ 6~
The results obtained ~re shown in Table 13 belot~.
The OCR engine was not
able to convert this image.
TEST EXA~IPL~ 8 (~1-2 Assay)
GRi~ l, GRA-~i-3 and GRA-~-4 obtained in REFERENCE
EX~IPLE2 ab`ove ~ere serially diluted with PBS ~0.85% NaCQ)
to prepare samples.
~ nti-1~-2 serwn from National Institute o~ Genetic
Reseclrch and the above sample were mixed and incubated at 4~C
for 2 hours and a target ce]l correspondin~ to the anti-H-2 '
lS ser11m used was added -thereto. Spleen cell or lymph node cell
obtainel:l Erom BlO ~1-2b) and BlO-BR ~H-2k) mice by conven-
tionclL method were used as target cell. After washing the
cells wi-t'h PBS, complernent (rabbit) was added to the cell and
the~ cells ~lere incubclted at 37~C for l hour and stained with
0.2~. tryparL blue-~BS to deter1nine % cytotoxicity. The anti-
~1-2 seLum was used in a maximum dilution such that it showed'
'L~ l~ast 95~ cytotoxicity in the absence of GRA.
'1'he blocking e~ect by G~ was determined or systems
showr1 in Table l4 below.
~z
52~
Tablc 14
GR~:~ Anti-~-l-2 Serum, Concentratlon _arget Cells
GRA-I\I-lD-23 (anti-1-1-2~(k~, X80 BlOBR ~H-2k)
or spleen c:ells
D-32 (anti~ 2Dk), X300
GR~ 1-3 D-33 ~anti-~1-2~b),X600 B10 ~H-2b)
or, spleen cells
D-2 (anti-~l-2DU),X80
GRA-~l-4 D--23 , X80 B10 - BR ~h - 2k)
or lymph node cells
D-32 , X300
The results obtainecl are shown in Table 15.
Table lS
~ Cytotoxicity
l\nti-H-2 _ 11ilu-tion of SamT)le
GRASerum X2 X4 X8 X16 .Y32 X64 X128 X25h X512 0 *
I - - 13 14 12 13 14 13 14 13 14 13
T1-23 - 97 99 96 97 97 96 97 96 96 14
D-3Z - 95 95 95 95 94 9S 9S 96 95 13
II - - 19 :15l't 12 15 12 11 12 13 14
D- 33 - 9$ 98 99 99 98 99 99 99 99 15
. D-2 - 95 96 95 95 97 95 97 95 95 17
XII D-23 100 100 100100 ~ - 100 10
D32 99 99 99 99 - - - - - 99 10
Notes. GR~ GR~
GRA II GRA-~I- 3
20. GRA III: GR~ l-4
"*" indicates % cytoto~cicity when complement
, ~ alone was u~;ed.
. ~ .
- ~3 -
: .
..... .. . ..
From the results sho-~n in Table 15 a~ove it can be seen
that GR.~ l GR~-~1-3 and ~R.~ M-4 lack H-2.
TEST E~A~IPLE 9
C57BI./6 mouse was transplanted under S.C. with LLC ~2X1063
5 rom the same strain and after 6 days 1 ng (protein) of GRA-
~l-3 obtained in REFERENCE EXAMPLE 2;(d) was administered subcuta-
neou-jly. Thereafter administration ~as repeated for 4 days
once a day at the same dosage. The day next the inal administ-
r~tion tumor cells ~ere e~cised and weighed As a control
physiological saline aclministered animals WeTe used. Each
~est grollp comprised 5 animals.
~ s a result, average tumor weight of the control group
was ound to be about 500 mg~ In GRA ~1-3 administered group
3 showecl disappearance of tumor and two had average tumor
weigllt: of ahout 100 mg.
TEST EXrUMPLE 10
CS7BI/6 mouse was immu.nizecl subcutaneously with 1 ng
(prote:i.n) of GR.~ 3 obtained in REFERENCE EXAMPLE 2~d) once a day
or 3 days and on day 5 spleen cel.ls were collected from the
anilllal. to obtain effector cells. A mixture o~ the e~ector
cells and lewis lung carcinoma ~LLC) as target cell in a ratio
of E/l = 50.1 was preparecl and a portion (lx105) thereo~ was
transplan-ted to mouse o the same strain and Winn assay was
performed (J. Immunol. 86 p.228-239 (1961~).
.
Ihe results obtainecl are shown in Table 16 below.
Table 16
Day 20
Effcctor Tumor Gro-~th Rate ~) Mortality/
Cells ~y 15Day 17 Day 18 Day 19Day 20 Group
:
A 5.7 - 5.5 5.7 3.1 1.4 0/lO
~ 21.~ 39.5 37.1 55.~ 76.8
C 39.3 65.1 61.7 ~8.1 96 4 4/10
Notes: GTOUP A represents spleen cells -F~om GRA-M-3
. iminune mouse.
Group B repres_nts spleen cells -from normal
JnOuse .
Group C represents splcen cells rom mouse
af-ter 10 days from the transplantation o
1,1.(~ (lx106~.
. TuJnor Growth Rate was calculated according to
the fo:l:Lowing equation:
Tumor Grow~h Rate t%) = rA rN x loo
N
wher~in Th represents thc ~hi.ckness of the Eoot pad bn ~he side
where the tumor ~as transplanted and T~ represents the thick-
ness of the nornIal Eoot pad.
.Tl:SI` EXAMPLE 1:l
C3i.-I/lTie mouse was immllnized ~ith ~,5 ~g (protein) of
GI~A-~I-4 obtai.ned in RE~RNCE EXA~IPLE 2 (d) and 0.1 m~ioE ~reund's
Cotnplete Adjuvant at the sacrococcygeal portion. Af-ter two
.
I weeks lymph node cells wcrc collected by conventional method
" . and were usecl as responder cell for determining proliferative
,,;,,; .
,.......... . .
~ 5 -
.
.~ ' . ' . , .
., ~ ,
,, .
response by GRA-~1-4.
For this purpose, res1)onder cells (~x105) were incubated
in RPMI-1640 medium containing 15% FCS in the presence of
GRA-M-~ for 5 days. During final 18 hours of the incubation
pe1~iod 1 ilCi of 3H-thymidine ~3H-TdR) was added to the medium ..
and its incorporation into the cells l~as counted.
The results obtained are sho~Jn in Table 17 belo~.
Table 17
Concent-ration 3H-TdE~ IncoTporation
GR~ `(ng/mQ) _ (mean cpm ~ S.E.) S.I.
Con~ro] 0 5,302 1,761
0.5 7J903 + 1,290 1.5
l 10,076 ~ 936 l.9
Experimellt 5 10,686 ~ 429 2.0
-10,615 ~ l,270 2,0
l'i ~0 . g,565 ~ 19 1.6
Note: S.I. indicates stimula.tion index in terms of
Experiment/Control.
TESr FX~MPLE 12
Delayed type hypersensitivity (DTH) response o~ C3~1/He
.20 normal mous~, X5563 im1llune mouse aJId MH].3~ immune mouse
obtained in.lthe same manrler as in EX~IPI.E 5, and GI~-M-~
immune mouse and GRA-M-5 immune mouse obtained in the same
manner as in TESI EXl~lPIJE ll was determined by foot pad
reaction (FPR).. That is, GRA or MMC-treated tumor cells
were challenged at the Eoot pad skin oE the hind leg of the
- ~6 -
.~ ' .
.. , , , . .. ~ .. . .,, .. , .. , ., ,... . ,, . . , . . ,~ ." . _,
~3S~6~
animal and swelling of tl~e foot pad 24 hours after the
challellge was determined. Degree of D'111 response ~as calcu-
lated by subtracting .the s~elling before challenge ~rom that
a~ter challenge ~lO 2 mm).
The results obtained are shown in Tables l8-22.
Table 18
__
_ ~Iean Foot Pad Increment rlO mm)
ChallengeNormal ~1ouseMHl34 Immune Mouse
-
l 2.8 13.6
2 12.~ 32.0
3 3.6 3.6
22.0
6.8 . 27.6
~otes: ~;roup l; syngeneic normal spleen lxlO6/20 ~Q
medium (~lanks solution)
~roup 2; ~1l34 cell lxlO6/20 ~Q medium
Croup 3; meclium 20 ~Q
:~ Gl~oup 4; GR~ , 0.8 ~g (prote1n)/20 ~Q mediwn
Group 5; " 0-4 ~g
Ta~le l?
_ ~leal1 Foot Pad IncreTnent (lO 2 mmj
Ch~].lenge Normal ~1ouse X5563 Immune ~1Ouse ~11-1134 Immune ~1ouse
; l 5.4 4.~ l.l
2 0.9 - 24.3
3 2.0 16.9
Notes: ~TOUp l; medium (~lan~s solution) 20 ~Q
~:roup 2j GR.~-~1-4, 4 ~g (protein)/20 ~Q medium
Group 3; GRA-~1-5,
- ~7 -
' . ,
~ - . ' - .
;26~
.
'I`able 20
~lean Foot Pad Increment rlO mm~
C _llen~ Normal ~tousc GRA~ 4, I~mune Mouse
1 1.7 -0.3
2 5.1 24.6
Notes: Group l; medium (Hanks solution) 20 ~Q
Croup 2; GRA-M-4s 4 ~g (protein~/20 ~Q medium
Table 21
Me~n Foot Pad Increment (10 2 mm)
Challe~ eNorrna:l MouseGRA~ ., Immune Mousë
_ _
~ -1.8 0.6
2 6~3 20.0
- 3 6.8 6.3
Notes: Group l; medium (Hanks solution) 20 ~Q
Group 2; GR~-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 o-~ G~-M-4 (hereinafter "C.P.")
Table 22
Me _ ~oot Pad Increment (10 2 mm)
h llenge Norma_ MouseGRA-~ 4, Immune ~louse
1 2.4 -1.0
~ I I 2.6 17.7
3 2.4 1.8
4 5.5 18.9
- ~8 -
Notes: Group l; medium (~1an~s solution) 20 ~Q
Group 2; GRA~ 4, 3.8 ~g (protein)/20 ~Q medium
Group 3, 3.S ~g (protein)/20 ~IQ medium of "C.P."
above
Group4; 3.8 ~ (protein) of GRA-~-4 and 3.8 ~g
(protein)/20 ~Q medium o "C.P."
REFERE CF. TESr
X5563 Immune mouse was obtained in the same manner as in
EXAM~'L~ 5. Spleen cells ~rom t}lis immune mouse were used as
effector cell and the effector cells (107) and tar~et cells
(X5563, 105~ were together transplanted to mouse of the same
strain. ~inn assay was performed in the sa~e manner as in
TES1' EXAMPLE :L0.
The -results obtained arc shown in Fig. 23, in which the
abscissa indicates days and the coordinate S]IO-~S Jnean tumor
size (cm ) -~ S~}.J~ and various rnarks represent the following.
o~ Group in W}liC}l effector cells were not added.
o~ o: Group in w]lich non-~reated effector cells were
adde(1.
~ : Group in which effector cells treated with
rabbit comple1nellt were added.
x~ x: Group in which e~fector cells treated with
anti-rhy l (New England Nuclear Co.,U.S.A.)
and rabbit complement were added.
o- o: Group in W]lic}1 effector cells treated with
anti-Lyt l (New England Nuclear Co. 7 U.S.A)
~52~
and rabbit complement were added.
u: Group in ~hich effector cells treated ~ith
anti-I.yt 2 (Nel~ England Nuclear Co., U.StA.)
- and rabbit csmplement were added.
From tile 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 kno~n that DTH response is mediated
by I.yt-l T~cells~ (J. _ p- ~lecl. 1~3 p.l534-3t3 ~1976)).
,~hile the invention has been described in detail
and ~ h reference to specific embodiment thereof, it will
be~ app.lrent to one skilled in the art that various changes
and modifications can be made therein without departing Erom
the spir1t and scope hereof.
,
- 50 -
., .
