JPS5859923A - Carcinostatic agent - Google Patents

Abstract

PURPOSE:To provide a carcinostatic agent containing cancer cell-disturbing lymphocyte (killer T-cell) specific to cancer cell-originated saccharide chain- relating antigen (GRA) as an active component. CONSTITUTION:The cell membrane component is removed from the GRA of the cultured cancer cell, transplanted cancer cell, spontaneously occured cancer cell, chemically or virally induced cancer cell, cancer cell originated from the operation tissue, etc. of man or animals, and the GRA is treated with lectin (e.g. peanut lectin, castor bean lectin, etc.) which bonds specifically to the terminal galactose. Lymphocytes are sensitized with said GRA to obtain killer T cells. The objective carcinostatic agent active specifically to cancer cells can be obtained by using said killer T-cell as an active component. The agent is preferably administered in the form of injection together with a carrier such as physiological saline water, etc. The concentration of the killer T-cell is preferably 10<5>-10<12>/ml, and the daily dose is 10<5>-10<12>/kg in 1-several doses.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is a novel anti-cancer agent, more specifically, it acts specifically on cancer cells that have a cell-derived glycan associated antigen (hereinafter referred to as 1"GRAJ), and 01 This invention relates to an anticancer agent containing cancer cytotoxic lymphocytes (hereinafter referred to as "killer T cells") as an active ingredient that destroy cancer cells.

Although immunocompetent cells, especially 1971 cells, which play a major role in cell-mediated immunity, undergo a rejection reaction against foreign cell antigens during transplantation immunity, this immunosuppression is not observed against cancer cells. weak.

Therefore, static cells are not destroyed and proliferate within the body, eventually causing the death of the tumor-bearing host.

The present inventor was conducting intensive research on the host's immune response system against cancer cells and its application to cancer treatment, and discovered that cancer cell-specific antigens, which are not found in differentiated normal cells, contain
We have discovered that there is a highly immunogenic GRA that acts as an immunogen on the host and establishes an immune response that is different from that of cancer cells.

When lymphocytes are sensitized to GRA, GRA
Killer T cells that act specifically against cancer cells that have cancer cells have been obtained, and when administered to a host, they recognize GRA and
The present invention has been completed based on the discovery that the present invention acts on and destroys cancer cells with A, and thus has extremely excellent effects in the treatment and prevention of cicadas.

Therefore, the present invention provides an anticancer agent containing killer T cells as an active ingredient.

Killer T cells of the present invention are produced, for example, by a method of sensitizing lymphocytes with GRA.

GRA used in this method includes human or animal cultured cancer cells, transplanted cancer cells, naturally occurring cancer cells, chemical substances and
It can be obtained from cancer cells containing GRA, such as virus-generated cancer cells and surgical tissue-derived cancer cells, as follows. That is, first, cell membrane components are separated from the cancer cells, and then lectins (J, B, 0
．． 250.

8518-8523 (1975); B10ehe
ffl.

Biophys Rea, Oomm, 6 2
, 1 4 4 (1975); 2, engineering mmu
nitaetsforch i 58.423-45
3 (1969); Br, J., Fixp, Fat
hol, 27.

228-256 (1946): Proc, Nat.
h.

Muscad, 8ci, USA, 75, A5, 2
215-'2219 (1978) = Biochem
istry 15, 196-2Q 4 (i 974
) ; Carbohydrate Reaeach
'51.107-118 (1976)), e.g. peanut lectin, castor bean (Rlcinua Com
mu-n1B) O can be easily obtained by treating with lectin, etc., binding to the lectin, and separating the cancer cell membrane components. This can be done by known methods.

More advantageously, for example, after homogenizing cancer cells in physiological saline or an appropriate buffer, the precipitated portion is collected by centrifugation or the like, and dissolved in physiological km water or a buffer using a solubilizing agent. This can be carried out by removing the supernatant by centrifugation or the like. The solubilizing agent used is
Various surfactants are generally known to be able to solubilize M cell removal, such as [Triton Examples include ionic surfactants and anionic surfactants such as sodium dodecyl sulfate (SD8).

GRA that binds to lectin can be separated from the microem components obtained in the above manner by conventional physicochemical or biochemical means that utilize the properties of the GRA. Examples of such methods include affinity chromatography using a column carrier containing lectin, immunoprecipitation using GRA antibodies, dialysis, RL filtration, electrophoresis, and glycoprotein precipitating agents such as polyethylene glycol and acetone. Examples include physical precipitation methods, etc., or methods in which these methods are appropriately combined. More advantageously, affinity chromatography using a column carrier containing lectin is preferred, and the column carrier can be easily obtained, for example, by immobilizing lectin on an insolubilized support. Here, the lectin can be immobilized on the insolubilized support according to a conventionally known biological material immobilization method. Among these, fixing methods using cyanogen bromide activated polysaccharide method, N-hydroxysuccinimide ester method, etc. are preferred. Among these, the cyanogen bromide-activated polysaccharide method is a method in which an insoluble support is treated with cyanogen bromide, and the obtained activated product is then coupled to a lectin under mild conditions to immobilize the lectin. When treating an insoluble support with cyanogen bromide, use a basic compound such as sodium hydroxide or sodium hydrogen carbonate to adjust the pH to 7.5-1.
17. Keep at room temperature under water, acetonate), 0.1M sodium bicarbonate buffer (...: 8.7), 0.01M phosphate buffer (P'l = 7.7), etc. 5-12
It is sufficient to perform homogeneous treatment for about 1 to 12 minutes in a solvent such as a buffer solution.

The amount of cyanogen bromide used with respect to the insoluble support is preferably approximately 1 isoelectric. Here, insoluble supports have low nonspecific adsorption to biological substances in general, high porosity, functional groups that can immobilize biological substances under mild conditions, and chemical and physical Any conventionally known insoluble support that is sufficiently stable can be used. For example, cellulose supports such as +f aminoethyl cellulose, carboxymethyl cellulose, bromoacetyl cellulose, lulose, p-anilinocellulose, cross-linked sequitran supports such as Sephadex, CM-Sephadex (manufactured by Pharmacia), Sepharose 2B, Examples include agarose-based supports such as Sepharose 4B and Sepharose 6B (manufactured by Pharmacia). When coupling the cyanogen bromide-activated support thus obtained with lectin, the cyanogen bromide-activated support was added at a ratio of 50% to lectin.
~80 times the weight, using a suitable solvent such as 0.1 mol sodium bicarbonate (containing 0.5 mol sodium chloride, -8
, 4) In an aqueous solution, usually about 0 to 40°C, preferably 2 to 40°C.
What is necessary is just to make it react at 8 degreeC for about 10-20 hours. In this way, a lectin-containing carrier for affinity chromatography is produced.

According to chromatography using an affinity chromatography carrier containing the lectin, the target GRA binds to the lectin in the carrier and is collected on the column. The column is then loaded with e.g. galactose,
Exchange reaction is carried out through a substance that binds to lectins such as biaxes having lactose at the end, oryza saccharai r, or highly concentrated salts, potassium thiocyanate aqueous solution,
GRA is dissociated and obtained by following an adsorption/separation agent (eluate) such as a cupric acid buffer.

The GRAs thus removed include lactose-terminated glycoproteins, glycopeptides, glycolipids and/or sugars.

There are no particular limitations on the lymphocytes, and any normal or tumor-bearing human or animal lymphocytes can be used. Specific examples include peripheral blood, bone marrow, lymph nodes, defibrillated cells, tonsils, and breast lymphocytes. Examples include those derived from etc. These lymphocytes can be isolated by physical or chemical methods, surface extraction methods, etc., and then subjected to the method of the present invention.

By G.R.A. Sensitization of lymphocytes is carried out using a medium containing GRA,
The lymphocytes are cultured for 1 to 10 days, preferably 2 to 7 days.

As the medium, various general nutrient media used for this type of cell culture can be used, such as RPMll.
640 medium, Eagle MEM medium, etc., to which human serum, bovine islet serum (yes), calf serum, horse serum, etc. are added are preferable. The amount of GRA added to the culture medium is usually 1 to 10 in terms of sugar per ix 10' lymphocytes/ml.
00 n97M1. % is preferably 1 to 500 n, 9/d.

Cultivation is carried out according to a conventional method, for example, at a temperature around -7.2°C or around 67°C.

Killer T cells obtained in this way contain T cell growth factor (T
In the above-mentioned medium containing CGF, T-1), the cells can be grown indefinitely while retaining their activity. In this case, the killer T cell clonin may be further selectively cultured by the usual limiting dilution method. Killer T cells can be stored stably for a long period of time, for example, by storing them in liquid nitrogen.

The anticancer drug containing killer T cells as an active ingredient obtained in this way is preferably made into an injection together with a carrier used in this type of blood product. The carrier is not particularly limited, but carriers that are isotonic with blood, particularly physiological saline, are preferred. In formulation, it is preferable that the killer T cells are sufficiently washed with physiological saline or the like to remove the above-mentioned medium, and then suspended in a carrier.

The killer T cell concentration in the preparation is not particularly limited, but
In general, 105 to 1012 pieces/ba is preferable. Furthermore, no toxicity was observed when killer T cells were administered at 1013 cells/mouse (intraperitoneally). The dosage varies depending on the type of disease, age, and sex, but it is usually 106 to 1012 pieces/unit/day.
It is preferable to administer the drug in several doses.

Next, Examples, Reference Examples, Test Examples, and Comparative Examples will be shown, but the present invention is not limited to these examples.

Reference example 1 (localization of GRA) ■ F engineering T' l1fk RL/ ri+7 (P N
Production of A-v engineering TO): Peanut lectin (PIM, manufactured by FiY) 10 Da to 0
．． 85% Na0z Noo,o IM-phosphate buffer (FJ4=7.2) 23! / dissolve in F
ITO (manufactured by Shikuma) 21V was dissolved in 0.5M-1 carbonate buffer (pH=9.0), and 0.5
1 to the above P11A buffer. After stirring for 2 hours at the source, the mixture was separated using Sephadex G25 (10x300x, manufactured by Pharmacia) and the first peak was collected. g/
p ratio = 1.0 ■ GRA localization in various tightening cells: 1 x 166 various human cultured cancer cells were treated with 0.85% NaC.
t0.05M-) Lis-HCl buffer (p)i=7.2)
Wash by centrifugation 3 times at
Allow to react in silence for 0 minutes. 0-85 fb N after reaction completion
After washing the cells three times with 01M phosphate buffer (...=7.2), the cells were placed on a glass slide and examined under a fluorescence microscope.

The results are shown in Table 1. The cancer cells tested were all known and were obtained from Daiichi Pathology, Niigata University School of Medicine.

Reference Example 2 (Preparation of GRA) ■ Production of insolubilized lectin (PNA-Sepharose): After thoroughly washing 3 g of ON Br-activated Sepharose 4B (manufactured by Pharmacia) with 1 mM HCt, 0.1 M-
Suspended in 200 xi of sodium hydrogen carbonate (pi-1 = 8.5), added 0.01 M phosphate buffer (P) 1 = 7.7) 5 at containing PNA201W, and heated at 25°C.
The mixture was reacted for 2 hours with occasional stirring to obtain PNA-Sepharose.

■ Preparation of GRA: Gel BT-i (Burkitt's lymphoma) cells 1.6x
108 pieces were washed 3 times with physiological saline, and 21r) Lidone
-100J (manufactured by Wako Tsumugi Yakuhin Co., Ltd.), 0.85 Ch NaCl, 2
mM-CaCjt2.2mM-MgC! t2 skin 01
M-) Add 30 g of Lis-HCl buffer (pH = 7.4),
Stir for 15 minutes at 4°C. Thereafter, the mixture was ultracentrifuged at 100,000 l for 2 hours. Of 28 ultracentrifuged supernatants, 14at
0.1 1 to 7 -100, 0,
85% NB-01%'1rnkA-CaC12,2
Tris-HCl buffer in mM-Mg0t2.

(p) i = 74), the PNA-A was plated using an Affinity chromatograph (φ0.5×1
1). After washing with the same buffer, 0.1M-lactose, 0.85% Na0t, 2mM-CaOt2.2m
M-MgCl2.0.1 Chidoriton X-100 no 0
．． Elute with 01'M-Tris-HCl buffer (pH = 7.4), and remove the bathing part with Q, 85% Nal, t, 2m
M-Mg012.2mM-CaOt20-01M0-
GRA solution 17 was dialyzed for 48 hours with 0l hydrochloric acid buffer.
Get m1. Folin the amount of protein and sugar of this
-As a result of measurement by the Lowry method and the phenol-sulfuric acid method, the protein amount was 644 μg and the sugar amount was 120 μg. Hereinafter, this will be referred to as "GRA-1J."

After washing 1010 lot C, H mouse breast cancer cells IX with physiological saline 6 times, 2%) IJt-ton X-100
, Q, 85% Nl! LOZ s 2mM-CaOt
Add 50ml of 2mM-Mg0t200.01M-1-Lis-HCl buffer (pH=7.4) and incubate at 4°C for 50 minutes.
Stir for 0 minutes. It was ultracentrifuged for 2 hours at u 10 n, o 00 x y, and the supernatant was diluted with 0.85 h of NaC! .

2mM-0aOt2, 2mM-MgC12
f) Dialyze overnight against 0.01 M Tris-Salt #buffer (pi-1=7.4). This dialysis fluid is mersible -OX ultra
-fi'1ters (manufactured by Millibore) to 6d, of which 1- is 0.005%) Liton X-
100, (3, B 5 @ Na0212mM-
CaO4z, 2rnM-MgO7-2(1) Tris-
Reference Example 2 equilibrated with hydrochloric acid buffer (SI=7.4)
-■ PNA-Sepharose affinity chromatography (
ψ0.5×2cm). ! Wash thoroughly with rs1 mild solution, 0.1 M-lactose, 0.85% Na0.
t, 2mM-0aOt2, 2mM-Mg0t2
, 0-0 0 5 To ) 'J To
7X -100 of 0.01 M Tris-Salt # was bathed in Seed buffer (-27.4) and the eluate was 0.85
% NaO4, 2mM-CaOt2.2mM-MgCl
2 of 0.01 M Tris-HCl buffer (pH=7.
Dialyze for 48 hours in step 4) to obtain GRAfl solution 2d. The protein content of this product was 156 μg and 94 μg per violet. This is hereinafter referred to as "GRA-M-1".

Reference Example 5 (Preparation of TCGII') Nippon Dell's defibrated JJkg obtained from Nippon Zorai Mates Co., Ltd. was removed and RPM Knee 164o medium (Florabora)
Wash twice with IJ-Co., Ltd.). The cells were filtered using a mesh (manufactured by Millibore, 150 mesh), and the cells were filtered using a heavy centrifugation method (
With a specific gravity of 1.076), 2t of lymphocytes of 2×10'/l/l are obtained. The lymphocytes were washed 3 times with RPM Nee 1640 medium, and 5 x 10 cells/cell were washed with the above medium containing 10% Fo8.
Adjust to 111t and incubate at 37°C in a carbon dioxide incubator.
Let stand for a while.

Collect supernatant lymphocytes and perform FC! 81 liters of the above medium
Adjust to 10' pieces/txl. Indomethacin (manufactured by Sigma) 1 pg/lit and PHA-P (manufactured by Difco) 0.2% were added, and the mixture was heated at 57°C in a carbon dioxide gas incubator.
Incubate for 48 hours. Centrifugation (3000x, 9°1
0 min), collect the supernatant, and pass it through a Millipore filter (0,2 min).
TC! GF
Get 27.

Reference Example 4 (Preparation of lymphocytes) ■ Human peripheral blood lymphocytes N'N liquid 501 obtained by collecting heparinized blood from healthy adults was centrifuged using "Ficoll Pack" (manufactured by Pharmacia Asia Panasonic), and peripheral blood lymphocytes were collected. Obtain balls 5x107i14.

(2) Mouse splenic lymphocytes C, H7'He mouse (S 6W') bladder viscera was removed and washed twice with RPM Knee 1 (54Q medium. Loosen with a syringe needle and then placed in a stainless mesh ( No. 100) Filter with a trowel to remove large debris. After washing the filtered cells with the above medium (2), centrifuge at 1200 x g for 10 minutes.
Obtain ×10' Ill IJ spheres.

Example 1 GRA-1 obtained in Reference Example 2-■-Ge 1 (with protein 14
0μg/lri, sugar content 7.5μg/ILl) in RPM knee 1 with Fe125% so that the final amount was 1.000 times.
Dilute with 640 medium to prepare sensitization medium.

5 x 10' 151 human peripheral blood lymphocytes obtained in Reference Example 4-■ are added to this Petri dish containing the sensitizing medium 5- and cultured at 57°C for 2 days. This is TCG720% obtained in Reference Example 3.
, ycsl 5chi containing RPM knee 1s4o = 5 more on ground
Cultivate for 1 day and collect 1×10 cells/ml of killer T cells 2.
Get 01. Hereinafter, this will be referred to as "GRA-1-ni-TJ".

Example 2 Reference Example 4-Mouse l obtained in ■! &klJ Npa ball FC
5X10'/! with 815% RPM Nee 1640 medium! I
GRA- prepared in / and obtained in Reference Example 2-■-101
Add M-1 to a final concentration of protein amount 1.5μj; l/Ml, sugar content 0.9μg/Ml,
The cells were cultured at 67°C for 2 days in a petri dish (60x60m, Falcon). Confirm cloning and further T
Cultivate for 4 days in RPM Nee 1640 medium containing 20 V/Y% of CGF (manufactured by Nippon Antibody Research Institute) in Fe815 to obtain 50 ml of 1×10′/rat Killer T Seij.

Hereinafter, this will be referred to as "GRA-M-1-, -TJ".

Example 3 The GRA-i-ni-TlQ''l solid obtained in Example 1 was dissolved in 10IIIl of physiological saline to prepare an injection.

Test Example 1 1 μl of GRA-1-T obtained in Example 1 was placed on a microplate (manufactured by Falcon) and allowed to stand at room temperature for 15 minutes. FC for this! 8 (Yapco Co., Ltd. 6') and let stand at room temperature for 30 minutes.
5% NaCt added [1,01M phosphoric acid buffer (pH=
7.2) Add 5 pi and centrifuge the plate for 5 minutes at 600 rpm+. Invert the plate and remove the unreacted 5
RBCN was removed, and staining # (PuIJ Leant Cressil Blue, manufactured by Merck & Co., Ltd.) was added to stain lymphocytes to check for positive rosette formation. As a result, more than 98% showed positive rosette formation (T-cells). - Test Example 2 Specific Cancer Cell Intoxication Activity 1 The following five cell lines with different GRA positive rates among the cells in Reference Example Table 1 were used as target human cells.

Target cytolytic rot 1, BT-1 (Burkitt's lymphoma) 2, Daudi (1) 3, NATO-Rin (Ken cancer) 4 MKN-45 (1) 5 MOLT (T-cell leukemia) target eel cell 5
Centrifuge at 800 rpm for 5 minutes to overlay 104 cells/well on a microplate (manufactured by Falcon). Next, GRA-1-4-T4X1034× obtained in Example 1
Gently add 10 liters and incubate for 1 hour.

The damaging activity was determined by the degree of plaque formation as follows.

Significant anti-inflammatory activity is observed.

+: Accept l.

±: l is slightly observed.

-: l is not recognized.

As a control, the experiment was performed using unsensitized human peripheral blood lymphocytes obtained in exactly the same manner as in Example 1 except that GRA was not used. The results are shown in Table 2.

From Table 2, it is clear that the killer T cells extracted by the method of the present invention have a strong, GRA-specific, small-talk disorder activity.

Below is the blank space Table 2 Table 1ot Same target cancer cells as above B1-3,2X 10
'rBx105 cells (cell ratio 5:1) for FC81
Mixed culture is carried out in 5 pieces of RPMI-164Q medium.

After 1 hour, the number of remaining cells was counted and the damage rate was calculated using the following formula.

The results are shown in Table 6.

(Table 6 1/) (Injury rate was determined in the same manner except that the ratio of GRA in lj, 1- to -T and target clastocytes was 5:6. The results are shown in Table 4.

Table 4 Test Example 3 0, H/He Spontaneous Breast Cancer-bearing Mouse in Example 2
t4sf,:3x10'10 of aRh-u-1-x-T
．． 3111/mouse was subcutaneously administered at 51al/W every other day. On day 10, the lesion was excised and the following pathological findings were obtained.

As shown in Fig. 12, lymphocytes infiltrated into the cancer cells and destruction of the tumor area was observed, and from Fig. 13, calcification of the tumor area was also observed. The antitumor properties of the cells were clearly observed.

Comparative Example 1 A case in which cancer cells themselves were used as a specific antigen instead of GRA in the method of the present invention will be shown below.

In Example 1, BT-1 and Da were used instead of GRAO.
udi, KATO-1- and MKN-45 lX105
Cancer cell-sensitized lymphocytes were obtained in exactly the same manner except that cells/petri dish were used.

The cytotoxic activity of these lymphocytes was investigated in the same manner as in Test Example 2-(A1). The results are shown in Table 5.

As shown in Table 5, no cytotoxic activity was observed in the lymphocytes obtained.

Table 5

[Brief explanation of the drawing]

Figure 1 is a photograph of Daudi@i cells, Figure 2 is a photograph showing plaque formation due to -T on GRA-1- of the same cancer cells, Figure 5 is a photograph of NATO-1 cancer cells, and Figure 4 is a photograph of the same cancer cells. Photographs showing plaque formation due to -T on Gl A-1- of cancer cells - Figure 5 is a photograph of BT-1 cicada cells, and Figure 6 shows plaque formation due to -T on GRA-1- of the same mesenchymal cells. photograph,
Figure 7 is a photograph of MKN-45 eel cells, Figure 8 is a photograph showing plaque formation due to -T on GRA-1- of isolytic cells.
FIG. 9 is a photograph of MOLT@ cells, and FIG. 10 is a photograph showing plaque formation by -T on GRA-i − of the same cancer cell.
Figure 11 is a photograph of BT-1 cancer cells treated with a mixture of naive human peripheral blood lymphocytes, Figures 12 and 16 are when GRA-M-1-T was administered to tumor-bearing mice. This is a photograph of the static cell structure of . Deployment Ceremony Japan Antibody Research Institute Co., Ltd.

Claims (1)

[Scope of Claims] ill@An anticancer agent containing @fm cytotoxic lymphocytes specific for cell-derived sugar chain-related antigens as an active ingredient. (21) The anticancer agent according to claim 1, wherein the cancer cytotoxic lymphocytes are obtained by sensitizing lymphocytes with cancer cell-derived sugar chain-related antigens. (3) The sugar chain-related antigen is The anticancer agent according to claim 1 or 2, which is a static cell membrane component whose terminal end binds to a lectin that specifically binds to lactose.