DD221917A5 - METHOD FOR THE PRODUCTION OF CANCER CYTOTOXIC LYMPHOCYTES - Google Patents

Abstract

The aim of the invention is the development of anti-cancer drugs. According to the invention, the method is to sensitize lymphocytes to a glycated antigen derived from cancer cells, wherein the antigen is a cancer cell membrane component that combines with a lectin that binds specifically to a terminal galactose or incomplete N-acetylgalactosamine.

Description

Process for the preparation of cancer cells - cytotoxic lymphocytes

APPLICATION OF THE INVENTION;

The invention relates to a process for the preparation

of cancer cell cytotoxic lymphocytes.

5 CHARACTERISTICS OF THE INVENTION ^:

However, immune-responsive effector cells, particularly T-lymphocytes, which play a major role in a cell-triggered immune response, cause graft rejection due to a foreign cell antigen does not produce appreciable or very limited immuno-inhibition against cancer cells. As a result, the cancer cells are not destroyed, but multiply in vivo and eventually cause the death of the cancerous carrier.

'-.is . ^: .--. ^:: '/ · ..'. : · '' · · '''..."· .- .-" ... ' ^: ': "'" ^! ': -. ";.

ΐ: '.- ' • ^; - ': - ::.' λ- '^;'''·':;.'.; ·. · ^: Λ ".' - 2 -. ; '. .. - .- "". '''. ·.

However, the existing mechanism is not completely clear and there are various studies

'' to get closer to the type of material basis that occurs in such a system. For example, cell surface markers on mouse leukemias or embryonic carcinoma cells are made using lectins such as Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA) in Biochem. Biophys. Res. Comm. > 9v (2) 448-455 (1979), Ibid. 96 (4) 1547-1553 (1980), J. Biochem. 89 473-481 (1981) and Ce_nj_8 183-191, September 1979 ".

Insofar as is known, no attempts have so far, however, been Undertaken to new lymphocytes available to: provide, can specifically fight the cancer cells, and the | present anti-cancer agent using a Immunansprechung of lymphocytes.

20 25

OBJECT OF THE INVENTION:

Thorough research on the immune response of the carrier to cancer cells and their use in the treatment of cancer have now shown that in a cancer cell specific antigen that is not found in differentiated normal lines, GRA is present * which acts as an immunogen for the carrier and a very high Has immunogenicity that causes an immune response that is specific to cancer cells. It has further been found that when GRA is used to sensitize lymphocytes, one obtains killer cells that specifically act on GRA-containing cancer cells, and that upon administration of the killer cells to a carrier, these cells. Detecting GRA and affecting / destroying GRA-containing cancer cells and, thus, achieving a very good effect in the treatment and prevention of cancer

PRESENTATION OF THE INVENTION OF THE INVENTION; The invention relates to a process for the preparation of. Cancer cell rcytotoxic lymphocytes.

The GRA used in the above method is obtained from GRA-containing human or animal cancer cells, for example, cancer cells / transplanted cancer cells, spontaneously occurring cancer cells, chemical substances or virus-induced cancer cells, or cancer cells which have been surgically removed from tissues using following Ver. · '. ^: drive. ''/'.-''?' ^: ..'. ' ^: '"' .y \. '"'" . , ''. · .V '''.'

One cell membrane component is segregated from cancer cells of the aforementioned kind and treated with a lectin which specifically binds with terminal galactose or terminal N-acetylgalactosamine, "where GRA can be readily separated with the lectin (see JBC, 250, 8518 Biochem., Biophys. Res. Comm., Bz, 144 (1575); Z. Immunol. Research., U8, 423-433 (1966); Brv J. Exp. Pathol., 2_7, 228-236 (1975); Proc. Nath Acad., U.S.A., TSr No. 5, 2215-2219 (1978); Biochemistry, ry, 196-204 (1974); and Carbohydrate Research, 5, 107-118 (1976)). whereby GRA unites with the lectin and can be easily separated.

The separation of the cancer cell membrane components can be achieved in a simple, known manner, e.g. by a homogenization method and a solubilization method using a resolving agent. It is advantageous to use a method in which cancer cells are homogenized in saline or in a suitable buffer, a part of the precipitate is collected by, for example, centrifugal separation, and in saline or a buffer by means of a solvent

is dissolved, after which the supernatant then separated for example by centrifugal separation. As a dissolution agent, there can be used, for example, surfactants generally known to dissolve cell membranes, e.g. nonionic surfactants such as Triton X-100 - (manufactured by Wako Pure Chemical Industries Ltd.) NP-40 (manufactured by Shell Co., Ltd.), digitonin and urea, or anionic surfactants, z> B. Sodium dodecylsulfonate (SDS).

From the cell membrane components thus obtained, GRA capable of cleaving with lectin using conventional chemical or biochemical methods and making use of the properties of the lectin can be separated. Examples of such methods are affinity chromatography using a column carrier containing a lectin, an immunoprecipitation method using GRA antibody or the like, a dialysis method,

a filtration method, an electrophoresis method . or physical precipitation using

'; a sugar protein precipitant, eg, polyethylene glycol and acetone, or a combination of such methods. Particularly preferred is affinity chromatography using a column which is a lectin _; wherein the column support can be easily prepared by immobilizing lectin on an insoluble support. The immobilization of lectin on an insoluble carrier can be applied in a known manner, as used for the immobilization of biosubstances

will, perform. Of these methods, it is preferable to use the cyanobromide activating polysaccharide method and an ininitization method using N-hydroxysuccimide ester. The cyanobromide activating polysaccharide method is a method in which an insoluble carrier is treated with cyanobromide, and the resulting activated product j is then subjected to a coupling reaction with a lectin under mild conditions to immobilize the lectin. For example, in the treatment of the insoluble carrier with cyanogen bromide, basic compounds such as sodium hydroxide and sodium bicarbonate are used to adjust the pH to 7.5 to 12 and then the carrier is treated in a solvent.

15 sungsmittel, such as water, acetonitrile or a

buffer maintained at pH 7.5-12, e.g. 0.1M sodium bicarbonate buffer (pH about 8.7) and 0.01M phosphate buffer (pH about 7.7) at room temperature for about 1 to 12 minutes. In general, it is preferable to use an amount of cyanogen bromide equivalent to the insoluble carrier.

Any known insoluble support exhibiting low nonspecific adsorption to all biosubstances, which has a high porosity and which has a functional group capable of immobilizing biosubstances under mild conditions and which is sufficiently stable chemically and physically, can be used of the present invention.

Useful insoluble carriers are, for example, carriers made from cellulose, e.g.

  -. 7 -

 The GRA used in the above method is obtained from GRA-containing human or animal cancer cells, e.g. of culturing cancer cells, transplanted cancer cells, spontaneously occurring cancer cells, by chemical substances or by virus-induced cancer cells or cancer cells which have been surgically removed from tissues using the following methods. "

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TO cell metabolic components are separated from cancer cells of the aforementioned kind and treated with a lectin which specifically binds with terminal galactose or endogenous

, wherein GRA can be connected with the lectin easily separated continuous N-Äcetylgalactosämin-i · binds (s JBC, 250, 8518-8523 (1975);.. Biochem Biophys Res Comm, bz,...

144 (1975); 't. Immunology., 228, 423-433 (19j56) j _: Bf. J. Exp. Pathol., 2T., 228-236 (1946); Proc. Nath.

Acad. Be. USA, 75_, No. 5, 2215-2219 (1978); Biochemistry, 12 '196-204 (1974); and Carbohydrate Research, 5 ±, 107-118 (1976)), thus uniting GRA with the lectin

and can be easily separated. , '; _..- Λ ".; ... '.':;::... ·. \ \ '''''.''' - ''

The separation of the cancer cell membrane components can be achieved in a simple known manner, e.g. by a homogenization method and a solubilization method using a dissolving agent, it is advantageous to use a method in which, cancer cells are homogenized in physiological saline or in a suitable buffer, a part of the precipitate is collected by, for example, centrifuge separation, and in physiological saline or a buffer by means of a solvent

is dissolved, after which the supernatant then separated for example by centrifugal separation. As the dissolution agent, there may be used, for example, surface active agents generally known to dissolve cell membranes, for example, nonionic surfactants such as Triton X-100 (manufactured by Wako Pure Chemical Industries Ltd.), NP-40 (manufactured by Shell Co., Ltd.), digitonin and urea, or anionic surfactants such as sodium dodecyl .Agent _f (SDS).

From the cell membrane components thus obtained, GRA which is capable of reacting with lectin using conventional chemical or biochemical procedures can be prepared and by the properties of the lectin. _\: Makes use separate. Examples of such methods are a (& affinity chromatography using ',: [, a column carrier containing a lectin, an immunoprecipitation method using GRA antibody or the like, a dialysis method,

a gel filtration method, an electrophoresis method, or a physical precipitation using

a sugar protein precipitating agent, eg polyethy-; ν glycol and acetone; or a combination of such methods. Particularly preferred is ₍ affinity chromatography) using a column containing a lectin, whereby the column support can be easily prepared by immobilizing lectin on an insoluble support. Immobilization of lectin on an insoluble support can be carried out in known manner Way, as applied for the immunization of biosubstances

will, perform. Of these methods, preference is given to using the cyanobromide activating polysaccharide

r method and an immobilization method using N-hydroxysuccimide ester on. The Cyanobromidäktivierungs polysaccharide method is a method

in which an insoluble carrier is treated with cyanobromide; The resulting activated product is then subjected to a coupling reaction with a lectin under mild conditions, whereby the lectin is immobilized. For example, when treating the insoluble support with cyanogen bromide, basic compounds such as sodium hydroxide and sodium bicarbonate are used to adjust the pH to 7.5 to 12. and then treating the support in a solvent such as water, acetonitrile or a buffer maintained at pH 7.5 to 12, z, B. a 0 / .1M, sodium bicarbonate buffer (pH about 8.7) and 0.01M phosphate buffer (pH about 7.7) at room temperature for about 1 to 12 minutes. In general, it is preferable to use an amount of cyanogen bromide equivalent to the insoluble carrier; ,

% ·, ^R : ν "'-. , '· · ·. '::;'.'.-,.;';-;.";'."

Any known insoluble carrier which shows a low nonspecific adsorption to all biosubstances, which has a high porosity and which has a radioactivity

having a normal group capable of immobilizing / biosubstances under mild conditions and being chemically and physically sufficiently stable can be used in the present invention.

Useful / insoluble carriers are, for example, carriers; made of cellulose, e.g.

Amihoethylcellulose, carboxymethylcellulose, bromoacetylcellulose and p-anilino-cellulose, or a carrier crosslinked! Dextran, · e.g. Sephadex and CM-Sephadex (manufactured by Farmacia Corp.) and a carrier of agarose, e.g. Sepharose 2B, Sepharose 4B Y ^ and Sepharose 6B (manufactured by Farmacia Corp.).

In the coupling reaction of the cyanogen bromide activated carrier obtained as above, the cyanogen bromide activated carrier is used in an amount corresponding to 30 to 80 times that of the lectin, and the reaction is carried out in a suitable solvent such as 0.1 mol / l aqueous solution of sodium hydrogencarbonate (containing 0.5 mol / l sodium chloride; pH 8.4)? een 0 and 40 ⁰ C, preferably 2 and 8 ° C zv at a temperature for a period of about 10 bis..20 Hours through. In this way a lectin-containing support for affinity chromatography is obtained.

In chromatography using the lectin-containing carrier prepared as described above, the desired GRA combines with the lectin contained on the carrier and is retained in the column. Subsequently, substances which are capable of combination, for example with a lectin,

run through the column with an exchange reaction, or alternatively an adsorption separation (with an eluting solution), for example with a highly concentrated salt, an aqueous solution of potassium thiocyanate and a nitrate buffer to separate the coagulated GRA and win.

In the exchange reaction, substances capable of binding to a lectin when a carrier for affinity chromatography containing a galactose-binding lectin is used, for example, those that combine with a terminal galactose-binding lectin, for example Galactose, disaccharides containing terminal galactose, and oligosaccharides containing a complete galactose. Examples of substances capable of combining with a lectin, when as carrier for affinity chromatography, an N-Acetylgalaktos-. ; amine-binding lectin is used are those which could bind S- to a lectin which binds to a terminal N-acetylgalactosamine, eg N-acetylgalactosamine, disaccharides containing a terminal N-acetylgalactosamine and oligosaccharides containing a terminal N- ^'-;; _#; i''galactosamine^-; acetyl -... · ". ^r - '' - ' _: . '"-.";: ··'; / '

The GRA thus obtained contains glycoprotein containing a galactose and / or N-acetylgalactosamine end group, glycolipids and / or saccharides.

If desired, the GRA produced in this way can be lyophilized and also purified in the usual way using customary separation methods. For example, such GRA preparations isolated with a galactose binding lectin can be subjected to a separation method using an N-acetylgalactosamine

Lectin and GRA, which was then isolated with a lectin containing N-acetylgalactosamine, can be treated by a separation method using a galactose-binding lectin.

The lymphocytes used here are not critical; and all lymphocytes of normal or cancerous'_; Humans or animals can be used. For example, lymphocytes obtained from peripheral blood, bone marrow, lymph nodes, spleen, tonsils, or thymus gland. These lymphocytes are isolated by physical or chemical methods, by a surface roughening method or by the same method. _ /; ";;. ;;; \ .; ^:

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  Sensitization of lymphocytes with GRA is carried out by placing the lymphocytes on a culture medium containing GRA for a period of 1 to 10 days,

1, preferably 2 to 7 days, cultured.

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As the culture medium for use in sensitizing the lymphocytes, there may be used various conventional nutrient media usually used for such; Cell cultivations are used. Preferred examples include RMPI 1'6 ^; 40 medium _for Eagle MEM culture, etc., htimanserum, fetal calf serum (FCS), calf serum, horse serum or the like

: has been. The amount of GRA added to the culture is generally 1 to 1,000 ng / ml and preferably 1 to

: -. 13 -

500'ng / ml, calculated as the amount of sugar · per 1 χ 10 / ml

T. virgin trivhen.

lymphocytes

The cultivation is in the usual way, ζ.?, At. a pH of 7.2 and at a temperature of about ': 37 ^e C.

The killer cells according to the invention thus prepared are essentially normal lymphocytes and have a GRA-specific cell-combating activity. For example, GRA-1-KT, one of the killer cells according to the invention, has properties which are found in human peripheral blood T cells and exhibits a cell-fighting activity. which is specific for cancer cells containing GRA, as shown in the examples below. 'V ^

Typical examples of the new killer cells according to the invention are GRA-1-KZ and GRA-M-1, which are prepared in the examples described below; the. These Kilierzellen are available from the applicant and deposited with ATCC.

The killer cells of the present invention can be freely multiplied using the aforementioned culture medium containing a T cell growth factor (TCGF, IL-2). In this case, selective cultivation of killer cell clones can be carried out by a conventional ultra-dilution method. The killer cells can be stable over long periods in /

be stored, for example, liquid nitrogen,

The GRA may be used singly as an active ingredient or may additionally be used in combination with other bactericidal agents and anti-cancer agents. Anticancer agents,

, which contain the GRA according to the invention as an active ingredient in any form, provided that they contain GRA in effective amounts. Generally, the agent is used intravenously, subcutaneously, intradermally or intramuscularly in the form of a solution, suspension or emulsion , You can also produce it as a product and Tröckö then immediately ι before use by adding a suitable carrier in a liquid transfer. This liquid agent; For example, a suspending agent, e.g. Methylcellulose, an emulsifying agent, e.g. Lecithin, preservatives, e.g. Methyl p-hydroxybenzoate, stabilizers and the like, as far as these have no adverse effect on the immunization function

in humans or animals. Buffers can also be used. be included.

Suitable aqueous carriers are, for example, a physiological saline solution, and a suitable nonaqueous carrier may be, for example, a vegetable oil,

- eg sesame oil, a mineral oil, ζ.B; Paraffin, or an animal oil, eg squallene, or else propylene glycol. To increase the immunological effect, it is possible to incorporate a suitable adjuvant. Suitable adjuvants are, for example, Freunds ¹ s V Complete Adjuvants, saponin in animals and aluminum hydroxide in humans.

The anti-cancer agent of the present invention may be administered once or repeatedly for prolonged periods to a cancer patient for the treatment of cancer, or it may be administered to someone who is susceptible to cancer, thereby preventing the cancer. ·

Since XiDe ₀ (mouse, intraperitoneal) of GRA is at least ν 500 mg / kg, calculated as the amount of sugar, the anti-cancer agent according to the invention shows a low: tokenicity and can therefore be used within a wide dosage range administer. Although the concentration of GRA in the anticancer agent according to

not critical to the invention, it is preferred to use it in an amount of 0 "001 to 100 μg / ml, calculated as the amount of the sugar. With regard to the

Dosage of the anti-cancer agent is generally preferred in the U25, the agent in an amount of 0.001 to; ': Share to administer, but depends on this dosage of the degree of disease, the age and sex of the patient 1.000 ug / kg / day at one time or in.verschiedenen arrival..

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The anti-cancer agent thus prepared, which is the

Kilierzellen as an active ingredient is preferably as an injectable solution in combination with carriers / in the manufacture of blood medicines. Find application used. The carriers used here are not critical, but preference is given to carriers,

which have the same tonicity as blood. Especially, physiological saline solution is preferable. In the preparation of the agent is preferably washed the; Killer cells sufficiently with a physiological Koch-TO salt solution or the like to remove the aforementioned culture medium, and then it is then ν / '^

The concentration of killer cells in the agent is not particularly limited, but is preferably

             - ·. / V- ··· .-, · ».. ·. Q -.

wise. between 10 and 10 per milliliter. If the killer lines are administered intraperitoneally at a dose of TO per mouse, no toxicity is noted. Although the dose of the anti-cancer agent of the present invention depends on the degree of the disease, the age and the sex of the patient, it is preferable that the agent is preferably administered in a dose of 10 to

10 / kg / day at once or in various ^{proportions:;} _. '' ^: ·,. '; administered ..; ^:: ; '} -;', ' ^[ .' , - /. ' , v --- · '''- ^: ' 2s ^. ^: y - [ ';; ·. "· .... ^{'::;:;.'..,.";} ; ^.'.-: - "'i' - ..-'- ','<The following examples, reference examples, experimental '.. Ν ν Step Example ieι and Ve'rgleichsbeispiele describe the ER; invention without to restrict it ..

- .17 -

Reference Example 1

(I) -A production of FITC-labeled lectin.

(PNA-FITC)

10 mg peanut lectin (PNA), manufactured by EY Co.) was dissolved in 2 ml of an O, 01 M phosphate buffer

AQ Of ⁸ ? % NaCl (pH 7/2) dissolved. In 1 ml of a 0.5M ^w Kydrogenkarbpnatpuffers (pH 9.0) 2 mg FITC (manufactured by Sigma Laboratories Ine.) Was dissolved and a 0.5 ml portion of the resulting solution was added to the previously prepared PNA buffer .; The mixture was stirred for 2 hours at room temperature and then separated on Sephadex G25 (10 mm mm 300 mm, manufactured by · Farmäcia Corp.). The initial peak was collected, FITC / PNA ratio: 1.0.

20. (D-B Preparation of FITC-labeled lectin

• (DBA-FITC)

'. , ^ . · '' .. ''. - '' ....

In the same manner as in (D-A above, DBA-FITC was obtained by using DBA produced by EY Co. EITC / DBA ratio »0.9.

(1) -C Soybean agglutinin-FiTCfTIFT-SBA) is available from Ey Co.

 FITC / SBA ratio »1.4.

(2) locality of GRA on different cancer

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(a) Human cultured cancer cells (1χ10) were washed three times with a 0.05 M tris-hydrochloric acid buffer containing

0/85% tiaCl (pH 7.2) was purified by centrifugal method and then 100 μM PNA-FITC, DBA-FITC ~ or SBA-FITC (200 μg / ml) was prepared and added according to (Ϊ). The mixture was allowed to stand at room temperature for 30 minutes to react. After completion of the reaction, the reaction mixture was washed three times with a 0.01 M phosphate buffer containing 0.85% NaCl (pH 7/2), and then the cells were placed on a glass plate and examined under a fluorescence microscope.

The results are shown in Table 1. The * cancer cells are all well-known and used by First Pathology Laboratories * Medical Department of Niigata University,

1-9 Table 1

cancer cells (Neuroblastoma) positive Ver from (Neuroblastoma) relationship (Neuroblastoma) GRA (%) DBA SBA (Burkitt Lyinphoma) (Neuroblastoma) PNA ¹ ' ⁴ Räji (Burkitt Lyinphoma) (Neuroblastoma) 98.3 5.2 · Daujl (Burkitt Lymphoma) 93.1 O BT-1 (T-cells Lyinphoma 50.1 6.7 P-12 (T-line Leukemia) 44.3 4.8 MOLT (B-cell lymphoma) 0.6 5.3 Fujimaki (IgD myeloma). 19.2 10.0 ODA, (Lung cancer, squamous) 0.6 2 * 0 QG-56 (Lung cancer , squamous) 70.4 0.4 PC-I. '·': .. ' (Lung cancer ', adenocarclnoma) 78.4 0 PC-3 (Lung cancer> small cells) 77.1 0 QG-90 (Lung cancer / big cells) 68.0 0 PC-13 (Stomach cancer , por.) 17.0 0.1 \ · MK-2 (Magenkregs sig.) 63.7 0 KATO-III (Stomach cancer por.) 57.3 40.3 MKN-45 (Stomach cancer, adsq.) 1 # 0th 0.4 MKN-1 (Gastric cancer / TUBL) 4.6 0.1 MKN-28 (Gastric cancer, TUBL) 0.4 0 MKN-74 (Bladder cancer ca ·) 0.5 0 MGH-Ut ' (Bladder cancer approx.) 37.4 21.4 CT-2 (Bladder cancer approx.) 4.5 0 T-24 (Bladder cancer approx.) 14.6 1.0 NBT, 2 (Kidney cancer) 13.1 O NRC-12 (Kidney cancer) 23.9 0.6 KU-1 Kuramochi (ovarian cancer) 3.3 0 NB-1 80.0 1.7 YT-nu 20.9 0.5 TGW-nu-1 3.6 O TGW-nu-11 4.1 1.0 GOTO 2.0 o · 0.5

ITO (Embryonal carcinoma) "" 96.9 12.3 90.7 NEC-8 (Embryonalcarcinoma) 44.6 0 6.4 SCH (Choriacarbinoma; stomach) 14.6 3.1 GCH (Chriocarcinoma, uterus) 5.4 0 YN-I (Rhabdömyosarcoma) 5.7 1.7. mouse X 5563 (Plasmacytoma) 92.0 0 mouse MH134 (Hepatoma) 18.6 0

(b) Malignant tissue from cancer cells was passed through a 150 mesh stainless steel sieve

Receives a single cell suspension. After washing twice with 0.01Mv HCl-Hit (pH 7.4) containing ₂ mMol CaCl ₂ , ₂ mM MgCl _2, and 0.85% NaCl, 5x10 ⁵ cells were obtained

100 μΐ of FITÖ-PNA or FITC-DBA (200 μg / ml) were added to the cell suspension and the mixture was incubated at room temperature for 20 minutes. After washing three times with cold PBS v, the cells were incubated under a fluorescence microscope . , ter searches ./ .- ,; 'V --.- ^; ·. .- '' v- '..-. ·.;'.-;;::; / 'ϊ''Ο -:'. 'ι.' :: -... .-,;. -. - ..... -.- -; ';.'^; : ''.

The results are shown in Table 2. The malignant tissue of cancer patients was obtained from Kansai Medical University.

- 21 Table 2

GRA

No. Cancer patient tissue PNA DBA

1 stomach cancer 2 stomach cancer 3 stomach cancer 4 Stomach cancer · 5 stomach cancer stomach cancer 7 breast cancer 8th breast cancer 9 breast cancer 10 breast cancer 11 colon cancer 12 colon cancer 13 Esophaguskrebs 14 hepatoma ! • Note: "+" sign

♦ ·. , '''*

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 '': '+'. '. '"

11 colon cancer + +

2Ö: · Note: '' + 'denotes the GRA on the cell surface;

"-" means that GRA does not occur on the cell surface.

f Reference Example 2 "

/ ^:: 25 · ', ".;.."'. ^: ''''''''''.''.-.' ^: '''': Production ^ of ^ GRA

(1) -A production of immobilized lectin r (PNA-Sepharose)

'' "'".,' · * »'

3 g of CNBr-activated Sepharose 4B (manufactured by Farmacia Cörp.) Was thoroughly washed with 1 mmol of HCl and suspended in 200 ml of 0.1M sodium hydrogencarbonate (pH 8.5). To this was added 5 ml of a p, 01 M phosphate buffer (pH 8.5) containing 20 mg of PNA, and then the reaction was carried out at 25 ° C for 2 hours while stirring several times to obtain PNA-Sepharose.

- (D-B In the same way as in (D-A above, with

: except that DBA was used instead of PNA, DBA-Sepharose was obtained.

S (2) Production of GRA ·

(a) BT-1 (Burkitt Lymphoma) cells (1.3 × 10 ⁸ ) were washed 3 times with physiological saline and then 30 ml of 0.01M tris-hydrochloric acid buffer (pH 7.4) containing 2 % Triton X-100 (manufactured by Wako Pure Chemical Industries Ltd.), 0.85% NaCl, 2 mmol CaCl ₂ and 2 mmol MgCl. The mixture was stirred for 15 minutes at 4 ° C and then subjected to a centrifugal vacuum deposition at a rate of 100,000 g. From 28 ml of the supernatant liquid thus obtained, a 14 ml portion was passed through a column (diameter 0 _r 5, length 1 cm) for the affinity chromatography, the column being filled with PNA agarose beads (manufactured by Maruzen Co., Ltd .)

2Q which had previously been equilibrated with a tris-hydrochloric acid buffer (pH 7.4) containing 0.1% Triton X-100, 0.85% NaCl, 2 mmol CaCl 2 mmol and MgCl _{2 2} mg MgCl ₂ , After washing with the same buffer as used above was treated with a 0 / OTM-tris Salsäurepuffer (pH 7.4) containing 0, TM lactose, 0.85% NaCl, 2 mM CaCl _2/2 mM MgCl ₂ and P, 1 4 Triton X-100 eluted. The proportion thus eluted

was treated with a 0.1 M tris-hydrochloric acid buffer solution, dec. holding 0.85% NaCl, 2 mmol MgCl ₂ and 2% ethanol CaCl ₂ dialyzed for 48 hours to give 17 mg of a GRA-r solution. In this GRA solution, the amount of pro-

tein and the amount of sugar according to the Folin-Lowry method or according to the phenol-sulfuric acid method determined J these amounts were 644 ug or, 120 \ ig. This product is hereinafter referred to as "GRA-I"'*'

: -V (b) c3 / BE. Mouse breast cancer ~ ^MMT - ^{2 cells} × 10) were washed 3 × with physiological saline and 30 ml of 0.01M tris-hydrochloric acid buffer. solution (pH 7.4) containing 2% Triton X-100, 0.85% NaCl, 2% ethanol, CaCl ₂ and 2 mmol MgCl ₂ /. The

Mixture was stirred for 30 minutes at 4 ° C. Subsequently, the mixture of an ultracentrifuge separator would be at a rate of J 0.0.0 * 00 g for 2 hours

And the supernatant was dialyzed overnight with a solution of hydrochloric acid (pH 7.4) containing 0.85% NaCl _r 2 mmol CaCl ₂ and 2 mmol MgCl ₂ . The thus-dialyzed liquid was concentrated to 3 ml, and a 1 ml portion was passed through a column (diameter 0.5, length 2 cm) filled with the same PNA-Sepharose as used before and filled with tris Hydrochloric acid buffer (pH 7.4) containing 0.05% Triton X-TOO, 0.85% NaCl, 2 mmol CaCl ₂ and 2 mmol MgCl ₂ , was equilibrated, subjected to affinity chromatography. After thoroughly washing with the same buffer solution as before, elution was carried out

a Q, 1M tris-hydrochloric acid buffer solution (pH 7, -4) containing O, IM lactose, 0.85% NaCl, 2 mmol CaCl ₂ , 2 mmol MgCl _2, and 0.005% Triton X-100, and the thus eluted portion became then dialyzed for 48 hours with a 0.01M tris-hydrochloric acid buffer (pH 7.4) containing 0.85% NaCl, 2 mmol CaCl ₂ and 2 mmol MgCl ₂ . Kan received 2 ml of a GRA solution. In this GRA solution, the protein content was 156 μg and the sugar content was 94 μg. This solution is hereinafter referred to as "GRA-M-1". , "

(c) Approximately 120 g (wet weight) of KATO-III was homogenized in a Waring blender in 100 ml of PBS. After centrifuging at 100,000 g for one hour, the pellets were dissolved in 100 ml of a 2% Triton X-100 solution in 0.01M Tris-HCl buffer (pH 7.6) containing 0.15M NaCl. The supernatant solution obtained by 100,000 g centrifugation for one hour was added to a column of PNA-Sepharose '4B (0.8x15 cm) previously treated with 0.015% Trito X-100 in 0.01M Tris-HCl (pH 7.6) containing 0.15M NaCl was equilibrated. After washing with 50 ml of the buffer, GRA was eluted with the buffer containing 0.1M lactose. The eluted GRA was dialyzed against 0.85% NaCl, concentrated by Sephadex Pharmacia Co. and at

"25-2i3 ° C 'stored before use.'

The amounts of protein and sugar determined in the same manner as in (a) above were 2.0 mg and 0.8 mg, respectively. This product "is hereinafter referred to as" GRA-2 "," ·· ..

(d) As in (c), the following GRA samples were obtained:

material Table 3 - · Amount (g) GRA Zuckerge GRA · source protein content halt (mg) sample 33 (Mq) * '. 0/09 . - - BT-a 0.5 GRA-3 excised  '' .5, 0.5 GRA-4 breast cancer 24 0.25 \ 0.38 QG-46 26 0.6 0.54 GRA ^ 5 QG-90 29 1.0 0.45. GRA-6 Raji 200 0.78 28.4 GRA-7 MMT 14.4 11.3 0.07 GRA-M-2 85 0.06 0.35 GRA-M-3 MH 135th 25 0.65 0.23 GRA-M-4 X-5563 0.56 GRA-M-5 ^

(e) In the same manner as in (c), but using NKN-45 (about 29 g) in place of Kato-III and using DBA-Sepharose obtained according to (DB, instead of PNA-Sepharose 4B and under Elution with N-acetylgalactosamine received in it a GRA preparation with a protein content

''. Of JD, 03 mg and a sugar content of 0.01 mg. This will be referred to as GRA-8 below. · '

(f) * GRA-3 prepared according to (d) (5 ml) was applied to a DBA-Sepharose column and washed with Tris-HCl buffer (0.015% Triton X-100, 2 mmol MgCl ₂ , CaCl ₂ , 0.85% NaCl), eluting 4 ml fractions No. 1-1: 2. Fractions 1-3 are referred to as "GRA-3-A" and fractions 4-12 as "GRA-3-B". Then, the column was eluted with the same buffer but containing O / N, IM N-acetylgalosamine, to give fractions designated "GRA-β-C"

become. ·.

(g) SDS electrophoresis

Each of the above GRA preparations was subjected to electrophoresis using the method of Fairbanks et al: BIochemistry, Volume 10 ': pp. 2606, 1971 *

The results are shown in FIGS. 18 to 22.

In Figs. 18-19, numbers 1 to 5 denote: 1 ... standard; 2 ... GRa-M-3; 3 ... GRA-7; 4 ... GRA-1; and

In Fig. 20, numbers 1 to 4 denote: 1 ... standard; 2. ..GRA-M-2; 3 ... GRA r6; 4 ... GRA-fifth

'-V -: -' - ' ¹ . '.; -. '.' · '..'. ' ... ··: '. ·;

In Fig. 21, numbers 1 to 3 denote: 1 ... GRA-M-4; 2, GRA-M-S..; 3 ... Standard:

In Fig. 22, numbers 1 to 4 denote: 20 1 ... GRA-3; 2 ... GRA-3-A; 3 ... GRA-3-B, 4 ... GRA-3-G,

Fig. 18 shows the state of GRA after protein staining according to C.B.B. (Fairbanks et al: Biochemistry / Vol. 10, p. 2606, 1971).

, ';;':.:. - ^; , '.' Figures 19 to 21 show the state of GRA after treatment with a sugar color reaction according to the PAS method (RM Zacharius et al., Anal. Biochem., Volume 30, p.128 (1962)).

Fig. 22 is a schematic illustration of the results of staining according to the aforementioned C.B.B. method.

As standard, the following were from Biorad Lab. Calif .USA used substances:

K Dalton; myosin

11 6 Il ; B-Gälactosidase 92 / 5 Il ; phosphorylase 66 2 " ; BSA 45 Il ; ovalbumin 21 , 5 β ; Sojabohnentryps

Reference Example 3

(a) The Japanese monkey spleen (obtained from Japan Plymates Co., Ltd.) was surgically removed and washed twice with an RPMI-1640 culture medium (manufactured by Flow Laboratory Co., Ltd.) were filtered using mesh (manufactured by Millipore Ine * _f 150 mesh) and a heavy gravity centrifugal (specific gravity

, · '. '.' , , , , η

1.076) to obtain 2 1 of 2.times.10.sup.1 / ml lymphocytes. The lymphocytes thus obtained were _; Washed 3 times with an RPMI 1640 culture medium and

the number of lymphocytes was adjusted to 5 × 10 ⁷ / ml using the same medium as before containing - 10% FCS. Then they were allowed to stand in a carbon dioxide fermentation apparatus for 1 hour at 37 ^° C. The supernatant lymphocytes were recovered

3Ov ',''·. ... '^;:'; - "'.''· ·.

- 2 δ -

and the number of lymphocytes was adjusted to 1 × 10 / ml using the same culture medium as previously stated containing 1% FCS. Subsequently, 1; '\ ig / ml Indomethacin (manufactured by Sigma Laboratories Ine.) And 0.2% PHA-P ^ (manufactured by Dipco Co.) was added and then the cultivation was in a carbon dioxide gas fermentation device'.:. 48 hours at 37 ⁰ C performed. After conducting centrifuge separation (3,000 rpm) for 10 minutes, the resulting supernatant was collected and sterilized by filtering through a MiXlipore filter (manufactured by Millipore Ιηέ .., Ö, 2 μίη) and to obtain 2 1 TCGF.

(b) As a source of TCGF, the supernatant fluid of misplated cultured peripheral blood lymphocytes became

- used by'10 healthy donors. Non-contiguous lymphocytes manufactured by CF. (Conray, Ficol (Japan Immunoresearch Co.)) separated lymphocytes by adsorption onto plastic surfaces at 37 ° C for 1 h _:. in RPMI 1640 medium containing 1% FCS (1.5x10 ⁶ cells / ml) ^ and containing 0.2% PHA-P, INdomethacin (1ug / ml) and V: Huiman Ör cells ( BT-1J (1.5x10 ⁵ cells / ml), pretreated

- incubated with Mitomyciri C (50 μm / ml). After 48 hours, the supernatant culture was harvested and identified as TCGF

Source (H.Inoue et al., Scand. J. Immunol. 12, p. 149-145 (1980)).

Reference Example 4

5 (T) Human peripheral blood lymphocytes

50 ml of blood obtained from healthy adults or patients with various cancers by heparinization / were subjected to centrifugal separation using Ficol Pack (manufactured by Farmacia Japan Co., Ldt.) To obtain 5 × 10 peripheral blood lymphocytes. -

15 (2) mouse spleen lymphocytes.

The spleen of a C3H / He mouse & (male, 6w) was excised and twice with an RPMI-1640 culture medium

.- 30 -

washed. Then, the spleen was loosened by an injection needle and passed through a 100 mesh sieve to remove large portions The cells thus filtered were washed twice with the same culture medium as above and then centrifugally separated at a rate of 1200 rpm for 10 minutes to obtain 4 × 10 6 mm of spleen lymphocytes.

το ^: '''·,f:.'',.' .-. ';'^;'.''

λ.; '- ^: .-- "'; Example 1 "., · '''..''' -. ·. ''.''·' -.

GRA-1 (amount of protein 40 μg / ml of amount of sugar 7.5 μg / ml, obtained according to Reference Example 2 (2a))! were reduced to 1,000 times the original volume with.; RPMI 1640 culture medium containing 15% FCS, diluted / with a sensitizer culture medium.

medium. ,

20 ^: '' - '^'; . . '. ; '''·; , .. -;. , , , '· · ·

Human peripheral blood lymphocytes (5 × 10/5 ml) obtained in Reference Example 4 (1) were added to 5 ml of the sensitizing culture medium prepared as above and this was then placed in a laboratory dish

25 and cultured at 37 ° C. for 2 days, followed by further culture on an RPMI-1G4Ö culture medium containing 20% TCGF and 15 % FCS obtained according to Reference Example 3 for a further 5 days using 20 ml of a killer cell solution containing

1 χ TO ⁶ killer cells / ml, received. This is referred to below as GRA-1-KT. ·

,:, ':' Example 2 ·. · - '. ·. .-. '.. ^: · -. , , /. '

The mouse spleen lymphocytes obtained in Reference Example 4 (2) were adjusted to a number of 5 × 10 / ml using an RPMI-1640 culture medium containing 15% FCS. To this was added GRA-M-1, obtained according to Reference Example 2 (2b), so that the final product contained 1.5 μg / ml of protein and 0.9 μg / ml of sugar. A 5 ml portion of the obtained mixture was cultured in a laboratory dish (60 mm χ Xs mm, manufactured by Faldon Co.) at 37 ° C for 2 days. The formation of clones was observed. The portion was further stored in RPMI 1640 culture medium containing 15% FCS containing 20% by volume of TCGF (manufactured by Japan Immuno Research Laboratories Co., Ltd.).

cultured for 4 days, to obtain 50 ml of a KiI * lerzellenlösung containing 1 × 10 killer cells / ml. This is referred to below as GRA-M-I-K-T.

Example 3 '

Lyinphocyten (5x10) from peripheral blood of healthy donors were cultured in RPMI-1640 medium containing 40 ng / ml (Pröteingehalt) incubated from GRA-2 and 15% FCS at 37 ⁰ C. On the second day, the aforementioned human TCGF was added to the medium until its concentration reached 20% and incubation was continued for 3 days; to obtain killer cells, these are referred to as "GUA-2 KT" hereinafter. · '

Example 4 \ · ^-:: '. .- - '''. , ') '. . ' · Ν,; '

Killer cell preparations GRA-8-KT, GRA-3-AKT and GRA-3-CKT were prepared in the same manner as in Example 1 using fRA-8, GRA-3-A and GRA-3-C {protein content: 50 ng / ml / and TCGF / which was obtained in Reference Example 3- (b).

G3HE / mice (female 8 weeks old) were implanted intradermally with the same strain, and after 7 days the tumor was surgically excised. After an additional 7 days, X5563 cells (10) of the same strain were inoculated and mice resistant to inoculation were blotted as an immune gland.

The MilzzeHen the immune mice and C3H / He mice ^wur-: prepared those applied by usual methods.

Each lot of spleen cells (5x10 / well) was sensitized with 40 ng / ml (protein content) of GRA-M-5 in RPMI 1640 medium containing TS% FCS for 5 days to give the killer cell.

Killer cell preparations obtained from normal spleen cells are hereinafter referred to as "GRA-M-S-K-t-V, and those obtained from immune spleen cells are designated as" GRA-M-5K-T-2 ".

Example 6

Human peripheral blood lymphocytes (5x10) were suspended in 5 ml RPMi-1640 ^ medium, disfounded at 50 ng / ml (protein content) GRAH; 1 incubated at 37 ° C for 2 days. On the third day (!), The lymphocytes were transferred to the RPMI-1640 medium containing 10% serum from the lymphocyte donor and (100 to 100 ng / ml (protein content) of GRA-1, the incubation became further 5 Days continued to give the Klllerzellen preparations shown in Table 4 below.

· · '.'.-. 'j; , ^; , ^; - '''''' . , ..- ·. ''. ''. '"

':' · '-''·.' - $ ':' : '"Table 4'. -.-.

ν;. .-; / -ν. (;; .. ry :. ' .. : .; ^: ,...; ·; GRA-Kohte'ht! ation (ng / ml)

First / day | , Day 3 killer cells

0 6 GRA-1-K-T-1 3, 2 GRA-1-K-T-2 ³ / GRA-I-K-T · ^ 6 5 GRA-1-K-T-4 12 GRA-1-K-T-5 25 GRA-1-K-T-6 50 GRA-1-K-T-7 200 GRA-1-K-T-8

50 50

50

   -. ' '. ·; ' '' '. 50 '' 50

Example 7 '· y / r ^: '. , '; , . ". ''

Ia) Peripheral blood lymphocytes (PBL) of various cancer patients after surgery were sensitized with GRA-3 to obtain killer cell preparations. PBL (5x10) from: the patients were incubated in RPMI .1640 medium containing 50 ng / ml (protein content) of lRA-3 for 2 days and then incubated in RPMI 1640 medium.

, '' '·.' -: '' · '' * · '

holding 20% TCGF and 15% FCS another 5 days on receipt

The cell cells / shown in Table 5.

P-GRA D-GRA 5 ·: ·. killer cells table -   '·! . "/ 'Peripheral blood lymphocytes + · '' + + '.. - · ·'. + ".- - · .." f .. +. ' - -. + -. ' - .- ' Days after the GRA-3-K-T-1 GRA-3-K-T-2 GRA-3-K-T-3 GRA-3-K-T-4 GRA-3-K-T-5 GRA-3-K-T-6 cancer surgery 14 · ;; '35 ^':: 21 7 35 21 Stomach cancer Stomach cancer Breast cancer Breast cancer Breast cancer Stomach cancer

In the above table, P-GRA and D-GRA fed the locality of GRA, expressed as malignant tissue (obtained by, surgery) from cancer patients, of which

Had been collected in the same manner PBL, as in _re: ferenz example 1/2 (b) ,. P-GRA and D-GRA were obtained using FITC-PNA and FITG-DBA, respectively. The days following the operation indicate the time when BPL will follow the

Surgery had been collected.

(b) PBL (5x10), collected from breast cancer patients 21 days after surgery, was in RPMI 164O medium containing

.25 50 ng / ml (protein content) GRA-3 and 10% serum from the patients incubated for 7 days to sensitize the ^ BL and to obtain killer cell preparations. These are referred to below as GRA-3-K-T-7.

"'Net ·, ^-.'; .. · '' - '. , '': '-

· .: ... Example 8 '. · ~ · ... "· \ -: - ' ^: ·''-V,' ¹ '/''''

(i) GRA-M-1> obtained according to Reference Example 2 (2b) was diluted with physiological saline so S that the content of sugar was 1.0 μg / ml and ^f of protein was 1 ', 6 ixg / tal . In this way, an anticancer agent No. 1 was obtained;

(Ii) A cancerous tissue of C3H / He spontaneously developing breast cancer became sterile to a 5 mm cubic. 'See lumps cut and transplanted under the dorsal skin of each 10 C ₃ H / He mice of the same strain as above (7 weeks old, male). 7 days after the transplantation, the fixation and multiplication of the cancer was confirmed. Five of these mice received subcutaneously the anti-cancer agent No. 1 prepared according to (i) in a dose of 300 μΙ / day in a two-day interval. The remaining 5 mice were used as untreated control. Ten days after the first administration, the tumor was surgically removed and its mean weight measured. At the same time, a pathohistological examination was performed.

Tumor volume,

** m * ~ Φ *, m * · m * ~ m ^ m mm Φ- ~ mm »-k .. _% . \. _ _

25 treated group: 22.3 mm ³ (Fig. 14) control: 162.7 mm ³ (Fig. 15)

This means that the tumor decreased by 86.3%.

In the control group (FIG. 16), bird's egg-like cancerous bodies were formed, v / obei the nature of the tissue

corresponded to a mark-like .kanalförmigen cancer and a multiplication of the cancer cells over the entire fabric was determined. On the other hand, in the group treated with the remedy ^; · .-.) (Fig * Π) the cancerous cells, a sedimentation necrosis at the sites where the cancer cells had formed and a calcification and. Firosis was detected with only a very small amount of cancer cells left. This confirmed the anti-cancer properties of the anti-cancer drug of the present invention

'J? . ' : ',.'. , ''' ^: -.-- "7' .. ' ^: ''y.'''. · '' - .-: '· -. ··': ". '-''''

: GRft-1 - ^ - T ti μ1); # obtained according to Example 1, was added to a microplate ^ manufactured by Falcon Corp.) and allowed to stand at room temperature for 15 minutes i. Then, 4 μl of FCS (manufactured by Falcon Corp.) was added, and the mixture was allowed to stand at room temperature for 30 minutes. neuraminidase

^; '''·:·' - '' .. N '' · ·

treated sheep red blood cells (SRBC). '.''' . · · · ·. It was added to the 0.85% NaCl to a number of 1χ10 / ml, and 5 μΐ of a Ό / IM phosphate buffer (pH 7.2) was added and then the plate was placed in a centrifuge trap Subjected rate of 6,000 üpm for 5 minutes. Then the plate was turned over and un-exposed SRBC ^N was removed. An antibody solution (Brilliant Cresyl Blue, manufactured by Merck & Co.) was added to stain the lymphocytes and formed into a rosette

 At least 98% show rosette positivity (T cells).

Test Example · 2

e_cancer cells_ destroying__ "activity

(a) Of the cancer cells shown in Table 1, the following 5 cell strains having different GRA positivity ratios were used as human cancer cells to be controlled:

15 cancer cells to fight:

No. 1 BT-1 (Burkitt Lymphoma) No; 2 Daudi (Burkitt Lymphoma) Nr ν 3 KATO-III (Gastric cancer) No. 4 MKN-45 (Gastric cancer) No. 5 MOLT (T-cell Leukemia)

On a microplate (manufactured by Falcon Corp.) 5 χ 10 * of the cancer cells to be challenged per well was applied by centrifugal separation at a rate of 8,000 rpm for 5 minutes. Then carefully ι 4 χ 10 were per well of by

GRA-1-K-T obtained in Example 1 and then incubated for 1 hour. ·

ib. .. '' .. ''. ^; ,: '' V '·'"; V. '. .- ..' ·· 'The killing activity was based on the degree of

Congestion detected and rated as follows: ++ a significant kill activity is determined ': -

+ a killing activity is detected + a low killing activity is determined

/ ^;: / "' ^: ';; represents · '.-''' · '·''';' . "," - "A" kill activity can not be detected

''. ·.. '- ^: '':.''·';'.'· · · - ^; , · / .. · · .-

in a control group unsensitized human peripheral blood lymphocytes were _f that had been prepared in the same manner as in Example 1, but without using GRA used. The results are in

IS '·' ·. , Ta) MlIe / € 'shown: ... · ..' ..... ;. .. · ·. -. ; , '

Table 6 shows that the Killeir T cells formed according to the invention have a strong GIIA-specific activity.

Table 6

to be combated 3y GRA-Positiwer- evaluation the  '... ·. , : cancer cell , .-: ratio (%) chalking GRA-I-K-T- '    "Daudi (Fig. 7) 93.1 ... V; · .- ' ++ (Fig. 2). group KATO-III (Fig. 9) "57.3 ^: \ '· ..: ^; - + (Fig. 4) BT-1 i (Fig. ' . '·. "" - ++ _"(Fig. MKN-45 (Fig. / < .- * · ·. +. (Fig. MOLT (Fig. 0.6 - (Fig.1 oyy Kontröll- bt-i:.; ::. 50.1 - CFig.1 group

.- 40, -

(b) The same 'cancer cells to be controlled as used in (a) (3.2 × 10) were mixed with 8 × 10 ⁵ GRA-1-K-iT (cell ratio 5/1) and the cell mixture formed ( Total number 4 × 10) was cultured in RPMI 1640 medium containing 15% FCS After 1 hour, the number of remaining cells was counted and the inhibition ratio was calculated according to the following equation:

cytotoxicity (%)

Number of cells (1 _ ₍ after cultivation _{) χ} Number of cells before cultivation (4 χ ΙΟ ⁶ )

The results are shown in Table 7,

10

Table T Another cell after the Kul tivierung KZyto- rtoxizität. · ' 2.9 χ 10 * 28 to combat de cells Number <before cultivation 3.7 χ10 * 7.5 Daudi 4 x10 ⁶ 3.2 χ 10 ⁶ 20 KATO-III ; 4 X TO ⁶ 3.9 χ 10 * 2.5 BT-1 4 χ TO ⁶ 4.2 χ 10 * MKN-45 4 x10 ⁶ MOLT * _; ' I 4 XiO ⁶

15

(c) The procedure of (b) was repeated except that the mixing ratio of GRA-I-K-T :. was changed to 5/3 to be combated cancer cells. The results are shown in Table 4.

20

table

25

30

to combat de cells Number <before cultivation. ; XiO ⁶ other cells. after the cultivation. ⁶ 10 ⁶ Cy toxicity. (%). • Daudi 4 ⁶ 10 ⁶ 3.6 ⁶ 10 ⁶ 91 KATO-III 4 ⁶ 10 ⁶ 3.6 ⁶ 10 ⁶ • 24 BT-1. 4 ⁶ 10 ⁶ 1.4 ⁶ 10 ⁶ 65 MKN-45 4 ⁶ 10 ⁶ 3.7 ⁶ 10 ⁶ 7.2 MOLT 4 4.1 -3

Test Example 3 ';. :: '-'. "''. ' '.,' / '.; / v ':. ''.'":.

C3H / He spontaneously breast-cancerous mice received subcutaneously GRA-MT-KT-, obtained according to Example 2, in a dose of »2 χ 10/0 , 3 ml corn 3 times a week every other day .. After 10 days was the Focus taken out and examined. In this experiment, it is found that GRA-1 KT exhibits high binding activity towards Daudi, KATO-III and BT-1 but only low binding activity to MKN-45 and MOLT.

'^;','"''.''.''.''.' . · ^'..'. .- 'in Figure 12 is shown, found an infiltration of lymphocytes into the cancer cells instead, and a break-up of the tumor area was found out. ^{Q: AEL} 5 · ¹ ^ shows that calcification of Tumor surface {has taken place and it can be seen that the inventive cancer cells have an antitumor activity.

Comparative Example Ί

In this example, cancer cells were used per se as specific antigens instead of GRA for the method according to the invention. .

Cancer cell-sensitized lymphocytes were obtained in the same manner as in Example 1, except that instead of GRA, BT-1, Daudi, KATO-III or MkN-45 were used in an amount of 1 χ 10 per laboratory dish.

With these lymphocytes became the cytotoxic

10

Activity was examined in the same manner as in Test Example 2 (a). And the results are shown in Table 9. \,. '}' _.::: :: ^ \. '' ..: .- ^: '- _:: - [: '':.:;,' :

Table 9 shows that the lymphocytes prepared above have no cytotoxic activity '^''^' .. '' - '..' · '''...'.'..

Table 9

 C;

15

20

- 25

30

to be controlled cell

BT-1 Daudi KATO-III MKN-45

Cells used for lymphocyte sensitization

Daudi

KATO-III

MKN-45

Test Example 4 '.''/"Γΐ^ν; ^ ^: ;;'...;;;''

In the same manner as in Test Example 2- (b), the anticancer activity of GRA - (- K-T, GRA-3-A-K-T and GRA-S-C-K-T obtained in Example 4 was determined The results are shown in Table 10.

GRA-8-K-T Table 10 GRA-3-C-X-T 8.0 4.3 20 0 % Cytotoxicity 5.0 20 0 cell GRA-3-A-K ~ T KATO-III BT-1, MKN-45 MOLT 5,0 5,0 5,0 0

'^{"'; ';}·;..''' Test Example 5::..." '-:' ι

(C) the cytotoxicity of the killer cell preparations; hold according to Example 6 was determined by means of a Cr-dyeing test (J, Immunol., 122, 2527-2533 (1979)). To

50 50 μCi of radioactive Cr (Japan Isotope Association) was added to ΚΑΤΟ-ΊίI (2x10) and the cells were ί h at

Incubated at 37 ° C. in RPMI-1640 medium and then washed by centrifugation to give Cr-labeled target cells The killer cells (effector cells) (2 × 10 ⁶ ) were added to the target cells (1 × 10) (ratio

• E / T was 20/1) and the mixture was incubated for 4 h at 37 ^° C. in RPMI-1640 medium. The supernatant fluid was collected by centrifugation and the radioactive

^{r was} measured by a liquid scintillator, ν

The specific Cr release (%) corresponding to the cytolytic activity of the fluoroeles was determined according to the following equation:

¹ Specific 'CR _ (Release in Trial)' .- (Spontaneous Release)

Specific CR release (%)

, (Maximum Release) - (Spontaneous Release)

, ::. : :: . : - 45 - -. - '- V; , · V

The maximum release shows QIe radioactivity when all cells are resolved. V

The results are shown in Table 11:

• 5 ^; v 'V vv,' V - ^: '. '. : .... '. ';.;.

'-' ⁽ - ^: ';"' ^: V - V: VV Vv ' Table 11 V-" V. ,, _'. · '....,

  Killer cells Specific release

GRA-ll-Kr-T-2. 25

GRA-1-K-T-3 22

; /. ' ; .. GRA-1-K-T-4 · 'V'V .. V 20. '";".

15 GRA- ^ 1-K-T-5 22

GRA-1-K-T-6 25

GRA-1-K-T-7 .27

GRA-1-K-T-8 32

From the results in Table 11, it can be seen that TCGF and FCS have no relation to the induction of killer cells. :

(b) Cytotoxicity of GRA-2-KT obtained in Example 3 was determined in the same manner as in (a) above by means of the Cr-expressing test. The specific Cr release was 14.3% for a ⁵¹ Cr-labeled KATO-III as the target cell (E / T-20/1).

, - 46

Test Example · 6 ; ^: · * V. , '... ^; _: -ν. , -. ^: [..- ':''"

(a) Using KATO-III (E / T = 20/1), the cytotoxicity of killer cells obtained in Example * 7- (a) was determined in the same manner as in Test Example 2- (b).

The results obtained are shown in Table 12:

Table 12 % Cytotoxicity Kilier-Zelleri GRA-3-K-T-1 GRA-3-K-T-2 GRA-3-K-T-3 GRA-3-K-T-4 GRA-3-K-T-5 GRA-3-K-T-6 38.0 18.2 20.9 23.2 24.5 20.2

(b) The cytotoxicity of GRA-3-KT-7 obtained in Example 7- (b) was determined using Cr-KATO-III (E / T s = 29/1) as target cells in the same manner as in Test Example 5 was determined. The specific .Cr release of the killer cells was 25.5%.

Test Example 7

The cytotoxicity of killer cells obtained in Example 5 was determined by the Cr release test in the same manner as in Test Example 5. The target cells used were Cr-labeled X5563 cells. As a control, lymphocytes obtained in the same manner as in Example 5 except that the sensitization of the spleen cells had been carried out with 1x10 / well of

35 mitomycin C-treated X55C3 cells (5x10 / ml of

X5563 cells were treated with 50 μg / ml mitomycin C for 60 minutes) instead of GRA-M-5.

The results obtained are shown in Table 13:

Table 13 Cr release (%) 1 20: 1 1 0: 1 Specific ⁵¹ E / T ratio 7 4 0 6.3 20.6 1 0.0 ⁵ ' ⁷ 3.7 0.0 killer cells 40: GBArM-S-X-T-I. ' GRArM-S-K-T? Control ' 12, 18,: 'o,

  - ''; ; ''. '·' .- -

Test Example 8 <H-2 Assay

GRA-M-1, GRA-M-3 and GRA-M-4, obtained according to the reference

 · .- '·. ·. , , , , , ·. ·.

Example 2 was subjected to serial dilution with PBS (0.85% NaCl) to give samples

National Institute o £ Genetic Research anti-H2 serum and the above samples were mixed and incubated at 4 ^e C for 2 h and then target cells were added according to the anti-H-2 serum used. Spleen cells or lymph node cells obtained from BIO (H-2) and B10 * BR (H-2) mice by conventional methods were used as target cells. After washing the cells with PBS complement (rabbit) was added to the cells and the cells were incubated at 37 ⁰ C and Vh with 0.2 5Trypan blue-PBS the percent stained for determining cytotoxicity. The anti-H-2 serum was used at maximum dilution so that it exhibited at least 95% cytotoxicity in absence of GRA.

fs .

10

20 25 ^:

30 35

The blocking effect of GRA in the systems is shown in Table 14:

''''','":-.,; ·., Table 14 ' ^: ''''' - . ' ''".',.''

, GRA Anti-H-2 D-23 D-32 : Serum, concentration X80 X300 Target cells GRA-M-I D-33 D-2 (Anti-H-2K ^k), or. (Anti-H-2D ^K), X600 X80 BlOBR (H-2 ^k) splenic cells GRA-M-3 ^ D-23 D-32 (Anti-H-2K ^b ), or _K (anti-H-2D ^D ), X80 X300 BIO (H-zV spleen cells GRA-M-4 ·. ·: '.-' · - ^: '' · · · .- ': drunk. , ': -. '' » BlOvBR (h-2 ^k ) lymph nodes

The results are shown in Table 15.

: Table 15

D-23 X2 - \ cytotoxicity X4 X8 X16 X32 X64 • X128 XZ 56 X512 14 • * Anti-H-2 D-32 - Dilution of the sample 13 14 12 13 14 13 14 13 96 13 GRA serum Il. , - - 97 99 96 97 97 96 97 96 95 14 I D-33 - 95 95 95 95 94 95 95 96 13 13 D-2 - 19 15 14 Γ-12 15 12 11 12 .99 14 III D-23 · - 98 98,999; ^; 99 9SS 99 99 99 95 15 D32 - 95 96 95 95 97 95 97 95 100 17 Annotation ..... 100 100 100 100 - · -      - -  '/ " 99 10   , , ', "' '' ' 99 99 99 99 - - - 10 , '! ...'-. - y ' GRA I -..-: GRA-M-I - '' ,, '. '-y.'". ^; -. GRA II: GRA-M-3 ·. ·· GRA III: GRA-M-4 % cytotoxicity means, if only. tl * tt -

Complement was used.

From the results in Table 15, it can be seen that GRA-M-1; GRA-M-3 and GRA-M-4 have no H-2.

.: ':'.: Test Example 9 ·. ^ ''''.'.''"."'' · -Ί ^: '"

C57BL / 6 mice were tested under S.C. LLC (2x10) of the same strain was transplanted and after 6 days 1ng (protein) ~ GRA-M-3; obtained according to · Reference Example 2- (d), administered subcutaneously. This administration was repeated once daily in the same dosage for 4 days. The day after the last administration, the tumor cells were excised and weighed. As a control, freezes were used, to which only physiological saline had been administered. Each test group consisted of 5 animals.

_>> . , ':.: "·',;. - ^: '.;''''. · -'. '' ^: '·' · .."' ^: '; · ".. '' The result was found that the mean cancer weight in the control group was about 500 mg, in the GRA-M-3 treated group 3 showed a disappearance of the ttimore and two showed an average tumor incidence.

weight of 100mg. ; _v

: >> Test Example 10 / V ^V "-. 'V"-.' ^: '' ^: ';:'. ''.'"''".;'-''" ^: ''

"·.;.. <* ·. '/ ..'. · ';"':. _i ·; , .. ·. · - '··. ' _V ... ·;

, .: ''. * * * "'''' ^v r- ^ri "' ^: , ": J" ^: -.': '' .. 's? - .'"';'.'.''

ν C57BL / 6 mice were immunized subcutaneously with 1 ng (protein) GRA-M-3 obtained in Reference Example 2 (d) once daily for 3 days, and on the 5th day, spleen cells of

collected to the animals to obtain effector cells. , A mix of effector cells and Lewis lung carcinoma

(LLC) as target cells in a ratio of E / T = 50/1 was prepared and a part (1x10) thereof was transplanted into mice of the same strain and a VJinn assay was duarched (J. Immunol., 86, p 228-239 (1961)).

The results obtained are shown in Table 16.

Table- 16 Day 17 Day 18 Day 19 Day 20 Day 20 Mortali effector 5, 39, 65, - »- ui Ui,; /; ·., 5.7 37.1 61.7 3.1 55.4 98.1 1 76 96 / 4 Λ ., t / fruppe ' cell 0/10 4/10 4/10 A B C Growth rate of cancer (%) Day 15 5.7 21.4 39.3

Note: Group A is the spleen cells of GRA-M-3 immune

- a mouse;

Group B are the spleen cells v ^v on normal mice;

Group C are the spleen cells of mice,

10 days after transplantation of LLCCTxIO);

15 '../''("-..''··.;.' * · '. ^{; 1} p

The cancer growth rate was calculated according to the following equation:

  , , , T * * "T Cancer rate (%) = - χ 100

where T _{A is} the thickness of the foot on the side where the cancer was transplanted and T _{n is} the thickness of a normal sole.

2.5 test example 11 ; "

C3H / He mice were immunized with 4.5 μg protein GRA-M-4 obtained in Reference Example 2- (d) and 0.1 ml FreundVs Complete Adjuvant sacrococcygeal. After 2 weeks, lymph node cells were collected in the usual manner and used as responder cells for the determination of proliferative response by GRA-M-4.

For this purpose, responder cells (4x10) were incubated with RPMI 1640 medium containing 15% FCS in the presence of GRA-M-4 for 5 days. During the last 18 hours of

-. At the time of incubation ^ Ci of H-thymidine {H-TdR) was added to the medium and incorporation into cells was counted. ''. ... '. "./. · .. .. .. ·. ·. ·. ·: ..;. \ ./Vji:: ^; v''.·''Ν:

The results are shown in Table 17.

GRA-M-R

Table 17 .

 .. '·. ι

Konzentra 3-H-TdR incorporation

tion (nq / ml) (mean cpm + standard error)

control

attempt

0.5

5 20 40

5,302 i 1 _T 761

7,903 i 1,290

10,076 i 936

10,686 jf 429

10,615 i 1,270

8,565 + 1,419

1.9 2.0 2.0

Note: "SI" is the stimulus index expressed by experiment / control ^: .'fi '~': ':., . V./.:::::::::::::::::::::::::::::::::::::::::

 Test Example 12

Delayed hypersensitization (DTH) response of CSH / HE normal mice / X5563 immune mice and MH134 immune mice obtained in the same manner as in Example 5 and of GkA-M-4 immune mice and GRA-M-5 Immuno mice, obtained in the same way as in Test Example 11, were determined by the foot-reabsorption (FPR). For this purpose, GRA- or Mt'iC-treated tumor cells were introduced into the skin of the footpad in the

The hind leg of the animals entered and the swelling of the foot insole. 24'h after the treatment was found. The DTH stimulation was calculated by checking the swelling

'·''. . '', · '· -. '. "-2''^;:"'

before the treatment of the after treatment (10 mm) withdrawn. ''·; . ';_; , '. _; · ·: ·. ''. ·. '' ·

The results obtained are shown in tables. * / ·. : '. '.'. , : "" ....

-

table

, ';''. ·. '·>'. .-. /,. . ' , ·· _2 '

Medium foot growth (10 mm) Experiment Normal mSuse _; MH134 immune mice

12.4 13.6 ι; '. ;; : ' 3.6 32.0 2 3.2 3.6 3 6.8 22.0 4 27.6 5

Annotation; Group 1s syngenic normal spleen 1x10 / 20 μΐ-

· Medium. (Hanks solution) ' Group 2: MH134 cells 1x10 ⁶ / 20μ1 medium

Group 3: medium 20 μΐ

^ Group 4: GRA-M-4, 0.8 pg (protein) / 20 μ1 medium Group 5: GRA-M-4, 0.4 μg «

- 53 Table 19

middle Stockohlensraachsen ( 2 10 now) attempt normal mouse X5563 immune mouse MH1 34 immune mouse * '· -I "- · 2 '. 3 : · '. 5.4 ^: 0.9 2.0  "" --ν 4,3 '16,9 1.1 24.3

OK Note: Group 1; Medium (Hanks solution) '20μ1' .'.' ...

Group 2; GRA-M-4, 4 μg (protein) / 20 μl medium '' '× group 3; GRA-M-5 * "

15 "· Table 20

_ - ^. , ^ Ii ι ι- ι ι _{i ι} β _

, _β '. 1

·. , , " -2 '" ^:

Medium footfall (10 mm). Normal Mouse GRA-M-4 Immune Mouse Trial

; Note: .Group 1.; Medium (Hanks solution 20 μΐ

Group 2} GRA-M-4, 4 μg (protein) / 20 μΐ medium

25 \, '"",:: .- .. ' · ...

attempt table 21 - / ν '; · .. ..; ... • ² ) Medium Foot growth (10 mm) normal mouse GRA-M-4 immune mouse 30 -1/8 6.3 6.8 0.6 20.0 6.3

"Message: Group 1; Medium (Hanks solution) 20 11 35 Group 2; GRA-M-4, 4 μg (protein) / 20 μΐ medium

Group 3; 4μg (protein) / 20μΐ medium of

Part that was hindoked by a PNA column at the time of GRA-M-4 (hereinafter ¹¹ CP. ")

    '. ". table 22 middle     '-2 foot increase (10 min) attempt Nornialmaus GRA-M-4 immune mouse , - ι. '. ·; '. '' · 2 ';,.' 3-ν .; '". ; '. ' 2.4 2.6 2.4 5.5 17.7 • '. , ':;.' λ, 8 ·. '/' 18.9

Note: Group 1; Medium (Hanks Solution 20 ) xl

Group ^: GRA-M-4, 3.8 \ in (protein) / 20 μΐ medium

Group 3: 3.8 μg (protein) / 20 μΐ of CP. (please refer

above).

.Group 4: 3.8 \ ig (protein) of GRA-M-4 and 3.8 μσ (protein) / 20 μ 1 of medium CP.

reference test

X5563 immune mice were obtained in the same manner as in Example 5. The spleen cells of these immune mice were used as effector cells and the effector cells (10) and the. Target cells. (X5563, 10) were transplanted together on mice of the same strain. Then, a Winn assay was carried out in the same manner as in Test Example 10.

The results obtained are shown in Fig. 23 / in which the abscissa indicates the days and the coordinate indicates the mean cancer size (cm ³ ) ± standard error and in which the various labels have the following meaning:

© - - ~ ο: group, given no effector cells softer

were; ö --- ο: group, to which untreated effector ..

Cells were given;

\ 10 A- ' - vd ^: group to which effector cells coincide with Kanin-S ^ v; · Chen complement had been given

were? χ-r - x: group to which effector cells treated with Anti-Thy 1 (New England Nuclear Co., USA). were, and to which:. Given rabbit complement

D: group to the effector cells treated with Anti-Lyt 1 "(New England Nuclear Co., USA), and to the. Rabbit complement was given _λ ; :

B., ..... @; Group, to the effector cells treated with anti-Lyt '(New England Nuclear Co., USA) and to.

_t , which was given to rabbit complement. >''<.''

From the results of Fig. 23, it can be seen that Lyfl.Type T cells play an important role in the mechanism of in vivo tumor immunity effect.

, Furthermore, it is known that the DTH response is caused by Lyt-1 T cells (J. Exp. Med. 143, pp. 1543-39 (1976)).

Claims (8)

Invention claim; A method for producing cancer cell cytotoxic lymphocytes, thereby to sensitize lymphocytes to a cancer cell-derived antigen derived from cancer cellh, wherein the antigen is a cancer cell membrane component that binds to a lectin which is specifically associated with a terminal galactose or terminal N-acetylgalactosamine, in combination: .niejrt; : '- ..; "." · · · · 2. A method according to EPOint Λ, characterized in that the glyc-related antigen was obtained by preparing cancer cell membrane components from cancer cells by homogenization or solubilization, the cell membrane component: with a lectin which specifically forms with a terminal galactose or with terminal N-acetylgaiacetic acid a lectin-cell membrane component complex, treats, collects the resulting complex, which separates lectin from the complex r Γ and a lectin-free cell membrane component wins. · "; V ^{: V:} / '..:"'. '.; · ' ^V \ ; 3 "method according to item 2, characterized in that the lectin is a lectin which binds specifically to a terminal galactose selected from the group of peanut lectin, Ricinuscommunis lectin and soybean lectin - a lectin containing specifically with a terminal N-acetyl galactosmain selected from the group of dolichos bean agglutinin, braid orange lectin, helix pomatia lectin, lima bean lectin, soybean lectin and bauhinobis lectin; 4. Method according to item 3, characterized in that said lectin is peanut lectin or dolichosulphonagglutinin. 5. ' Method according to item 2, characterized in that the glycologically related antigen : $ '. isolated from a cancer cell membrane component, by first reacting it with a first lectin which binds specifically to independent galactose and then to a second lectin which specifically binds to terminal N-acetylgalactosamine. 6. Method according to point 5, characterized g e k e η η - It can be seen that the first lectin is peanut lectin and the second lectin is dolichosorbent agglutinin. ".": ". , .,. 7. A method according to any one of items 1 to 6, wherein the sensitization is carried out by incubating lymphocytes in a nutrient medium containing a cancer cell-derived glyc-related antigen _; cultivated during a period of 1 to 10 days. e. ^ Procedure according to point 7> thereby <g e ken η - " ^: '" ^: .. "^"' _; ν,:: z; \ &": ± ch η et" / that the amount of glycated antigen is 1 to 1000 mg / ml. 9. The method according to item 8, characterized in that culturing the lymphocytes at a pH of about 7.2 and a temperature of about 37 ° C. 10. Method according to item 9, characterized in that the nutrient medium RPMI 640 is a medium or an Eagle-MEM medium, the medium containing human serum, fetal calf serum, calf serum or horse serum. ύ ,, _ΜΙ , yes awesome drawings