JPS5859922A - Cancer cell-disturbing lymphocyte - Google Patents

Abstract

PURPOSE:To obtain the titled lymphocyte (killer T-cell) reactive specifically to cancer cells, by sensitizing a lymphocyte with a cancer cell-originated saccharide-chain- relating antigen (GRA). 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, 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. The GRA bonded with the lectin is separated therefrom, and the killer T-cell can be obtained by culturing lymphocyte in a medium containing said GRA at about 7.2 pH and 37 deg.C. The killer T-cell can be proliferated indefinitely in the above medium containing a T-cell prolifration factor, keeping the activity of the cell, and can be stored in liquefied nitrogen stably for a long perid. Representative examples of the lymphocyte are GRA-1-KT and GRA-M-1-KT.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to cancer cytotoxic lymphocytes (hereinafter referred to as "killer T cells")
More specifically, the present invention relates to killer T cells that act specifically on cancer cells having the cancer cell-derived sugar chain 4M, HA (referred to as GRAJ) and destroy the cancer cells.

Immune agent aI@, especially 7171, which is the main player in cell-mediated immunity
Although the cells readily reject foreign cell antigens during dream immunization, this immunosuppression is not observed or is weak against cancer cells.

In the body, cancer cells are not destroyed and Jlffit,
, ultimately leading to the death of the cancer-bearing host.

While conducting intensive research on the immune response of the host exposed to fig@ and its therapeutic application, it was discovered that in cancer cells, S-antigen, which is not found in differentiated normal cells, was found.
Immunogenicity acts as an immunogen on the host and establishes a specific immune response with 4 cells, resulting in %t/k GRA
It highlighted the existence of

And this Gl;jA1F! 'A killer that sensitizes lymphocytes and acts differently on cancer cells with GRA? They discovered that cells can be obtained and completed the present invention.

The killer T cell of the present invention recognizes GRA
It acts on cancer cells with GRA and destroys them, so it is effective in treating and preventing cancer. For example, it is produced by a method of sensitizing lymphocytes with GRA.

The GRAs conveniently used in this method include GRAs of human and animal cultured cancer cells, transplanted cancer cells, naturally occurring Nh cells, chemical/viral salivary cancer cells, and surgical tissue cells.
It can be superior to @l@ with , as follows. That is, first, the cancer membrane components are separated, and then a lectin (T, T,
B, 0.250 *8518-8523 (1975)
; BioChem.

Blophys Res, QOmm, 62. 14
4 (1975); 2, engineering
oh 138, 425-433 (19/) 9)
; Br, J., J.P., PathOl, '
i7.

228-236 (1946); Proc, Nat.
h, ACad.

Sci, USA, 75145.2215-2219(1
978) ; Blochemistr3' 13*
196-204 (1974); ca
rbohyarate Re5each.

51 107-118 (1976)], analogy (f, bi□
1. Nut lectin, castor bean (Rlcinus Oom)
munis) lectin, etc., and binds to the lectin and separates it.

1!1a@ Separation of membrane components can be done by, for example, the homogenate method, 1
This can be achieved by a known method such as a solubilization method using lli'TI@-ization/IJ. More advantageously, for example, cancer #1@menstrual fluid #: Weir water father is homogenized in a suitable slow @@ solution, the precipitated portion is collected using a centrifugal valve 4I, and this is soaked in fresh water or slow water. This can be carried out by dissolving 1# Keishin using a town bath kakeru and removing the supernatant by centrifugation or the like. As the OT solubilizing agent, there are various surfactants known to be able to solubilize cell membranes, such as "
Triton! -100J (manufactured by Wako Tsumugi Co., Ltd.), “1p-40
J (manufactured by Shell), nonionic surfactants such as dichitonin, urea, etc. Examples include anionic interfacial viscosity agents such as sodium sulfate (8DS).

Furthermore, the GRA that binds to lectin from the a m g bx obtained above can be separated by conventional physicochemical or biochemical means that utilize the properties of nuclear GRA. Examples of such means include affinity chromatography using a column carrier containing lectin, Gl'j
A-Immunoprecipitation method using antibodies etc., dialysis method, gel F1N4
Examples include methods such as electrophoresis, permeation, physical precipitation using a protein precipitant such as polyethylene glycol or acetate, or a combination of these methods as appropriate. More advantageously, affinity chromatography using a column carrier called lectin is preferred, and the column carrier can be easily obtained, for example, by immobilizing lectin on an insolubilized support. Here, immobilization of lectin on an insolubilized support is
This can be carried out according to conventionally known methods for immobilizing living organisms. Among these, cyanogen bromide-activated polysaccharide,
It is preferable to use an immobilization method such as the N-hydroxysuccinimide ester method. Among these, the cyanogen bromide-activated polysaccharide method is a method in which an insoluble support is treated with cyanogen bromide, and the resulting activated product is coupled to a lectin under relaxed conditions F to immobilize the lectin. When treating an insoluble support with cyanogen bromide, for example, use a monobasic compound such as sodium hydroxide or sodium deoxygenate and maintain the pi-17.5 to 12 at room temperature with water, acetonitrile, or 0.1 MIR sodium hydrogen acid buffer (-48
, 7), about 1 to 1 in a solvent such as a buffer with an rff of 7.5 to 12, such as 0.01 M phosphoric acid (-U7.7).
It is enough to process it for about 2 minutes and 11 minutes. Generally speaking, it is preferable to use approximately the same amount of cyanogen bromide for the insoluble support. In this case, the insoluble support has low non-component adsorption to biological substances in general, has high porosity, has monofunctional groups that can immobilize biological substances under relaxed conditions F, and has chemical and physical properties. Any reasonably stable insoluble support can be used. For example, cellulose supports such as aminoethylcellulose, oxymethylcellulose, zolomoacetylcellulose, p-anilinocellulose, crosslinked dextran thread supports such as Sephadex, CM-Sephadex (manufactured by Pharmacia), and 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 a lectin, the cyanogen bromide-activated support is used at a ratio of 60 to 80 times the lectin, and an appropriate solvent, preferably 0.1 molar sodium bicarbonate (containing 0.5 molar sodium chloride, -8.4) in aqueous solution, usually O to 4
The reaction may be carried out between 0''C species, preferably at 2 to 8°C for about 10 to 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. lactose,
Exchange reaction is carried out through two types of substances that bind to lectin, such as olissatucharide, which has lactose at the end, or high-grade 1, potassium thiocyanate water bath solution,
aRi through an adsorption/separation agent (eluent) such as boric acid buffer)! ! , ml.

The GRA thus obtained contains lactose protein, shelf peptide, mq quality and/or IR@
@ string.

There are no particular limitations on the lymphocytes, and any normal or tumor-bearing human or animal lymphocytes can be used. Specific examples include the terminal octopus, bone marrow, lymph nodes, PS, tonsils, thymus, etc. Examples include the origin. These lymphocytes can be isolated by physical or chemical fishing methods, surface membrane methods, or the like, and then subjected to the method of the present invention.

Sensitization of lymphocytes with GRA is carried out by culturing lymphocytes in 3M soil containing GRA) for 1 to 10 months, preferably for 2 to 7 days.

As a medium, you can use the general old Eiji 4iI medium used in this Hinoki no 4, @j@ culture, but for example, RPM Engineering 1640 medium, Eagle MFiM medium, etc., human serum, fetal bovine serum (PCB). It is preferable to use one containing , horse serum, etc. GRA that can be added to the culture medium
is usually 9'l for lymphocytes I x 106 @/mno,
1 to 11000 n/m as mt.

, 1-500 ng/ml is preferred for samurai.

＠Nyo is carried out according to the usual method, for example around 7.2 of the pilgrimage, at a temperature of around 67°C.

Killer T cells thus obtained can be grown indefinitely while retaining their activity in the above-mentioned medium containing TaN-enhancing 9fl factor (TQGiF, Engineering Lu). 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 killer T cells of the present invention produced in this way are 7978 cells (T cells) with normal lifespan and are unique in that they have a cytotoxic activity particularly S-circle against GRA. For example, one of the killer T cells established and isolated according to the present invention (GRA-
As shown in the test examples described below, in addition to the common properties of human peripheral blood T cells, i-KT) exhibits a strong cytotoxic activity specific to 8a@ having GRA.

As a representative example of this new killer T cell, the present inventor has
Ill i'! according to the examples described below. GRA-
ATCIC! An application is being made for its deposit.

It is preferable that the killer T cell of the present invention is made into an injection together with a carrier that has been used in this blood type agent.

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 concentration of killer T cells in the cell is not particularly limited, but is generally preferably 105 to 10121 cells/ml. Furthermore, no toxicity was observed when Killer T Sale was administered to 1013+ mice (intraperitoneally). The dosage depends on the severity of the disease,
Depends on age and gender, but usually 105-10'21
It is preferable to administer the drug in one to several doses per day.

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

Reference example 1 (localization of GRA)
Manufacturing: Peanut lectin (PNA%BY company #) 1° to 0-
0.01 M-phosphor of 85 qb aaoAt! I1
．． Dissolve in 2 tsl of bottle buffer solution (pH 7,2). Dissolve 2 das of F engineering (Sigma Co., Ltd. #) in 0.5M carbonic acid resistant liquid (p) I = 9.0) 1 #l/d,
5 ruler: Add to the above PNA cr) laxative solution. After stirring at room temperature for 2 hours, Sephadex G25 (10a
+ m x 300 m, Pharmacia #) and collect the first peak. K/P ratio = 1.0 ■ Various @! GRA localization in 1llA cells: Tanidan human cultured cancer MJ cells I
0.0 5 M - Tri Sue 4# Loose @g (
After washing with the same centrifugation method at tH = 7.2), the PNA-F to (200μfi/d>) obtained above was centrifuged at 100%
μ/μ/ml and react in a quiet manner for 30 minutes at room temperature. 0.85 sound NI after the reaction is completed! After washing three times with Lal's 0.01 M phosphate buffer (-=7.2), a@ was placed on a glass slide and examined under a fluorescence microscope.

The results are shown in Table 1. The 8 cells tested were all known and were obtained from Department of Pathology, School of Medicine, #Gata University.

Table 1 Reference Example 2 (manufactured by GRA A) ■ Production of insolubilized lectin (PNA-Sepharose) 4i
: 0NBr -> fs Sepharose 4B (7 Almacia [J 311 k1mia-klat), washed with light, 0.1M-*sodium hydrogen oxide (FJ-
1=8.5) 200ml K $ 114, F N
A 20m9'4! :fftr 0.01 M-Phosphoric acid 4 weak #g (pH=7.7) 5 ml was added to 7JO, and reacted at 25°C with occasional stirring for 2 hours to obtain PNA-
Obtain Sepharose.

■ GRA, Jll: (Il B?-1 (Burkitt's lymphoma) small @ 1.6
×108 pieces were washed 3 times with physiological saline, and υ1 tritoyX
-100J (Wamoto Pure Chemical Industries #ri, 0.851sacl,
0.01 M Tris salt of 2 mM-cacl, 2 mM-Mtqal, 1! i! Buffer! (p)l=7.
4) Add 30ml of waste and stir at 4"C for 15 minutes. Then, ultracentrifuge at 100,000XI for 2Q. Add 28rnl of ultracentrifugation supernatant to ζ14 at 417°0.1%.
Triton x-ioo, 0.85%N
a01. 2mM-aac et al., 2mM-Mg0l
*PNA-A equilibrated with Tris-HCl buffer (tJ (=7.4)) is applied to Loos Beads (Maruzen Co., Ltd. Affinity Chroma) (10.5 x 1 m). After washing with the same buffer, 0.1M-lactose, 0.85% Na□l,
'lmM-0&Ol, , 2mM-
Mg(V2, 0.19/X-1
0.01 M Tris-HCl buffer (pH=7
．． 4), elute S with 0-85% NtLO6,
2mM-M (<06@, 2mM-caolg
of 0.01 M-tris-ti
Dialyze with acid*#g for 48 hours and transfer to GRA bath g17ILt.
Superior. FOI in-
As a result of measurement using the Lowry method and the phenol-sulfuric acid method, the protein content was 644 μy/mt and 120 μy. This is hereinafter referred to as rGRA-IJ.

(mouth 1 03H mouse mammary cancer fine @ l
0, Q, 85% Na0J, 2mM-cacJ
, 2mM -J (Jl of 0.01M-Tris-Gas acid buffer f(tJ (=7.4))
Stir for 0 minutes. Then i o o, o o o x
Ii for 2 minutes rIJ14 centrifugation, and the supernatant was diluted with 0.85% N
acll, 2mM-cace, 2mM-M
0.01 M −) of gals (…=
7.4) overnight. 1g of this dialysis is processed.
mers1b1θ-OX [Jltra-fill, er
a (manufactured by Millibore) to 5dKI, and one of these was 0.005% Triton X-100, 0.85% Na□
l, 2mM-cacJg, 2mM-M
Tris-salt 112 of gCj12 (...= 7.4
) and subjected to PNA-Sepharose affinity chromatography (00,5×2cnL) of Reference Example 2-■, equilibrated with ). After thorough washing with the same buffer, 0.1M-lactose,
0.854r Na0J, 2mM-aacg,,
2mM-MtlolB, 0.005% Tri-7 to-10000,01M-Tri-41! Elute with both liquids (F) I = 7.4), and reduce the eluted portion to 0.85%.
NaC71! , 2mM -CaO#2.2mM
-MtqO et al.'s 0.01M-) Risu t1! # Dialyze GRA solution g2 with buffer solution (pi (=7.4) for 48 hours
Get ml. The protein content of this product was 156 μ9, and the protein content of Sugi 1 was 94 μg.

This is hereinafter referred to as jGRA-M-IJ.

Reference Example 3 (To( ) IF d4m) The pancreas of 4 Akebono was removed from Nippon Dell (obtained from Nippon Primeira Co., Ltd.) and washed twice with RPM Knee 1640je base ( ) O-Rad Laboratory Co., Ltd. M). Mesh (manufactured by Miliboa,
Lymphocytes were collected at 2x109/ml by specific centrifugation (specific gravity 1.076).
Go to *1. This lymphocyte is RPM knee 1640 @ pond 6
Wash twice and use the above medium containing 10% PO3 for 5×10711!
/ 1111 K A jar L, in a carbon dioxide incubator.
Incubate at 67°C for 1 hour and 14 hours.

Supernatant lymphocytes were collected and incubated in the above medium with 8i% FO.
i oa / RI K , Jl set. Ingemesacin (manufactured by Sigma) 1 μg/R1, X'Hk-P (Difco 11) 0.2 t was added and cultured at 37° C. for 48 hours in a carbon dioxide incubator. Centrifugation (3000X
g% 10 min), collect the supernatant, and pass through a Millipore filter (
0.2 μm, millipore III)
Obtained to GF2j1.

Reference example 4 (rA@ of lymphocytes) ■ Human peripheral blood lymphocytes 50 ml of blood obtained by heparinized blood collection from a healthy adult
“Ficoll Pack” (Pharmacia Zoyapan Co., Ltd. 11)
Centrifuge at 1) and collect 5 x 10' peripheral blood lymphocytes.

(2) The pancreas of mouse pancreatic lymphocytes c3H/1ie-rus ('J, sw) is removed and washed twice with RPMI-16404. Stainless steel mesh (1001) after loosening with a syringe needle! At P] I! Remove large debris. After washing the F-treated cells twice in the above-mentioned @ solution, centrifugation was performed at 1200 x gl for 0 minutes to obtain 4 x 10' ill of tile lymph 4.

-4! Reference example 2-G Riichi (GRA-i (protein 1 obtained in step 1)
404/ml, sugar 1k 7.5 t4/ILt) k
en 1'cs-i5soR to be 1.000 times
Dilute with PMI-1640 medium to prepare sensitization medium.

Add 75 ml of 5 x 106 human peripheral blood lymphocytes obtained in Reference Examples 4 to ① to a Petri dish containing 5 IIL of this sensitizing medium, and store at 37°C for 2 days. echo. This was cultured in Reference Example 3 for another 5 days in a RPM knee 1640 medium containing 15% of IP (3F3), and then
106 (IIA/lit killer T cell 20d is obtained.Hereinafter, this will be referred to as ``GRA-1--TJ''.

Example 2 Mouse mIIIJ lymphocytes obtained in Reference Example 4-■ were 7081
GRA-M obtained in Reference Example 2-■-(B) by fA splitting to 5X106/m/ at 5% RPM knee 1640f@ground.
-1 *, i concentration protein charge 1-5 ttjj /1t
il, D so that the sugar filter is 0.9μj9/ml,
5 ml of it was kept at 37℃ for 2 days in a petri dish (60 x 6
0 mm, Falcon). Confirm the presence of the resin and add TOGF (manufactured by Japan Antibody Research Institute) 20
V/V% Fe215% RPM Knee 1640
Cultivate J@ in the medium for 4 days to obtain 1x1Q'/m/killer T.
Obtain cell +50m/. Hereinafter, this will be referred to as “GRA-M-1-
- Referred to as TJ.

Test Example 1 1 μl of GRA-1-Ni-T, which was superior in Example 1, was placed on a microplate (manufactured by Falcon) and incubated at room temperature for 15 minutes. Add 4 μl of Fe2 (Gipco, $11) to this and leave it at room temperature for 60 minutes. I x 10' pieces/ml
K
4 (5RBcN) (7) 0.85%NaCeJ
J port 0.01 M-phosphorus #R buffer (...=7.2)
Add 5 μce and centrifuge the plate at 600 rpm for 5 minutes. The plate was inverted, unreacted 5RBON was removed, and a staining solution (Brilliant Cressil Blue, manufactured by Merck & Co., Ltd.) was added to stain lymphocytes to examine the longevity of rosette formation.

As a result, more than 98% of the cells showed positive rosette formation (T-cells).

Wipe+! $ Column 2 Temporal Cancer Cell @ Injurious Activity (A14) The following 5a cell lines having different GRA positive rates among the cells in Table 1 of the same reference were used as the target human cancer cells.

S 1% 1, BT-i (Purkitt's lymphoma) 2, Daud
i (tr)3, KATQ-11(
Gastric cancer) 4, MKN-45 (1) 5, MOLT (Tm! sexual leukemia) target cancer cell @5
Fill a microplate (Falcon Co., Ltd.) with X10'l well by centrifugation at 80 rpm for 5 minutes.

Next, 4x10 GRA-1-T obtained in Example 1
3+t! Gently incubate the wells for 1 hour.

Obstacle! ! F activity was determined by the degree of plaque formation as follows.

Ox: Significant #4I activity is observed.

+: Accept l.

Soil: A slight I is observed.

-, I is not recognized.

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

From Table 82, it is clear that the killer T cells produced by the method of the present invention have a strong cytotoxic activity with different GRA%.

Table 2 (b) Same mFFJ8m cell as in (a) above 3.2
-T '8X to GRA-1- for 10' pieces
105 cells (cell ratio 5:1), a total of 4 x 10' cells, were mixed and cultured in RPM knee 164 (lF!) with 15% FQB for 1 hour.The number of remaining fIi!d@ was counted and the failure rate was determined. was calculated using the lower manure method.

The results are shown in Table 3.

Table 3 Hi1 (In DI, the disability rate was determined in the same manner except that the ratio of GRA-'1- to -T and target cancer cells was 5:3. The results are shown in Table 4.

Blank space below □ Table 4 Test Example 6 03H/kiθ Spontaneous breast cancer-bearing mice were treated with 4 GRA-M-l-T in Example 2.3X10'10.3El
/mouse was subcutaneously administered 3 times/W every other day.

On the 10th day, the autostatic lesion was excised and the following pathological findings were obtained.

As shown in Figure 12, lymphocytes infiltrated into the tumor, destruction of the tumor area was observed, and calcification of the ulcer area was also observed from @ 13 +m, indicating that the killer T cell target of the present invention Anti-axial tumor properties 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 GRA.
u(l i, KATo-II and MKN-45
Cancer 1 was prepared in the same manner except that 105 pieces/Petri dish were used.
1al@sensitized lymphocytes were obtained.

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

From Table 5, no cell 4# activity was observed in the lymphocytes obtained.

Table 5

[Brief explanation of drawings]

Figure 1 is a photograph of Daudi cancer cells, Figure 2 is a photograph showing plaque formation due to -T on GRA-i- in the 8th orifice, Figure 3 is a photograph of xATo-111 Kusunoki cells, and Figure 4 is a photograph of Daudi cancer cells. Photograph showing plaque formation due to -T on GRA-1- of cancer cells! Figure 5 is a photograph of BT-1 cancer cells, Figure 6 is a photograph showing plaque formation due to -T on GFiA-i- of the same cancer cells,
Figure 7 is a photo of the MKN-45 Gantsumugi gun, and No. 8 is the same gun gun.
A photograph showing plaque formation due to -T' on GFjA-1- of @, Fig. 9 is a photograph of MoLT Kusunoki @, Fig. 10 is a photograph showing plaque formation due to -T on GRA-1- of the same cancer cell, Figure 11 is a photograph of BT-1@ cells treated with a mixture of naive human peripheral blood lymphocytes, and Figures 12 and 13 are when GRA-M-i-T was administered to tumor-bearing mice. This is a photograph of cancer cell tissue. Above Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 11 Figure 12 Figure 13

Claims (1)

[Scope of Claims] 1. Cancer cell lymphocytes specific for cancer cell at-chain antigens. 2. The range of TlfIfX obtained by sensitizing lymphocytes with cancer a@Yume sugar fa-related antigens. 3. The 51al cytopathic lymphocyte according to claim 1, wherein the sugar chain-related antigen is a @#1illi membrane component whose terminal end binds to a lectin that specifically binds to lactose.