JPS6098000A - Monoclonal antibody of anti-human cancer - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody of anti-human cancer to be reacted with glycolipid of fucosyllactosamine type derived from human cancerous cell shown by the formula I (Gal is galactose, GlcNAC is N-acetylglucosamine, Fuc is fucose, Glc is glucose, Cer is ceramide, n is 1 or 2), not to be reacted with lacto-fucopentaosyl (III) ceramide shown by the formula II. USE:A diagnosticum or remedy for human cancer. PREPARATION:Mammalian immunocyte and myeloid cell immunized with difucosyl neolacto-morhexaosylceramide and/or trifucosyl neolacto-noroctaosylceramide, glycolipid of fusocyllactosamine type derived from human cancerous cell shown by the formula I are subjected to cell fusion in the presence of polyethylene glycol, etc. to give a hybridoma, which is cloned, cultivated, to give a monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel monoclonal antibody that selectively and specifically reacts with a specific 7-cosyl lactosamine type glycolipid derived from human invaginating cells.

In the present specification, carbohydrates, lipids, their binding modes, etc. are indicated according to IUPAC-IUB, the common name or customary name in the field of this glaze.

It is known that the Q-turns of complex carbohydrates on the cell surface change as cells become cancerous, and the causes of these changes in sugar chains are thought to be defective glycan synthesis of glycolipids and unique sugar chain structures. It has been suggested that the appearance of glycolipids with

The present inventor has been conducting various studies on the above-mentioned matters for some time, and in the process, it was discovered that glycolipids having a specific sugar chain structure were separated from human cancer cells, and that the sugar chain structure was unique to human cancer cells. We discovered that this is an antigenic structure that is commonly expressed in cells, and filed a patent application (%Application No. 111857).
-198433). That is, as human cancer cell Fi tumor formation progresses, the following general formula (1)% formula% (11 [wherein, Gat is galactose, GteNAcilt,
N-acetylglucosamine, Fue is fucose, Ol
e is glucose, Cer is ceramide, and n is l or 2].
e2nLe6 , n = 2 (D mo(D'j:l
"V3vll" Fuc, indicated as nLc8). In addition, some types of camphor cells have already produced lactofocopentaosyl (1) ceramide (Laeto-fueop@ntaosyl).
(1) e @ramld・;111FucnLe4
) is known to accumulate (J, Dial
, Chem, 246.1192-12004- (i7i)':l. In contrast, monofucosyl neolacto-norhexaosylceramide (monofuocyl neolacto-norhexaosylceramide) was recently extracted from normal human cells.
-norhexao*ylcsramlds+; V
3FucnLc6), monofucosyl neolactonenoroctaosylceramide (monofuao@yl
neolaato-noroetaoayl-co
ramido; ■l”FuenLcg) and zofcosyl neolactone 4-octaosylceramide (di
fucosyl n@olacto-noroeta
osylcc+ramide; vSVIFuc2nL
cB) was isolated [J, Blol.

Chem, 257.14865-14874 (198
2)).

Based on these results, we conducted intensive research to obtain antibodies that are specifically reactive with the unique sugar chain structures that appear with canceration, and found that a specific S-lipid derived from human aneurysm cells and a selected specific antibody. The present invention was completed by successfully obtaining monoclonal antibodies YSa, ysa, YSe, and YSf, which react with each other and do not react with normal cells.

The present invention is produced from a hybridoma obtained by fusion of immune cells of a mammalian animal and bone marrow lung cells after immunization with the above-mentioned 13Fuc2nLc6 and/or M3V''V13FuclnLeg,
A fucosyllactosamine-type glycolipid derived from l”!1/
"Fne2nLc6 and/or l1ljV" VIFFu
The present invention provides a monoclonal antibody that specifically reacts with e3nLc4.

Incidentally, several types of monoclonal antibodies produced for the above purpose have already been reported, such as anti-5
SEA-1 [formerly ochem, Blophym.

Res, Commun., 100, 1578-15.
86 (t98i); Proe, Natl, Aead
, Scl, U8A 75 * 5565-5569 (
1978); Nature 292.156-158
(1981)'3, wGus 29, ZWG13, ZW
G14, ZWG l 11 [Arch, Bioehe
m, old ophym-217+647-651 (1982
)'), 538 F12.538FB [Arch, B
ioehem, Blophys, 220 * 318-
320 (1983)], VEP8, VEP9
(I8.ur, J.

Immunol, 13, 306-312 (1983))
, M, -1 [Blood 61.1020-1023(
1983):) etc. are known. However, all of these monoclonal antibodies have an X-determining group (Ga
tβl −+ 4 [F u eαl−+3 ] Gt
eNAC). Therefore, since these antibodies react with any fucosyllactosamine-type glycolipids containing normal cell components, it is not possible to distinguish between these glycolipids.

The monoclonal antibody of the present invention is 13V derived from human tumor cells.
3Fue2nLc6 and/or amount 3VB■'Fue3n
It is produced by fusing immune cells of a sentinel mammal immunized with Lel with bone marrow lung cells to generate a hybridoma, from which a monoclonal antibody having the above-mentioned properties is isolated.

In the present invention, the hybridoma is prepared by a method known per se, for example, Nature 256.495-497 (
1975) and its modifications (J, Exp, M.
ed, l 5011008-1019 (1979)]
It is done according to iniquity.

Immunogen 13''FuelnLc6 and/or Ham3
■s■3Fuc3nLeg is obtained from various cultured human cells expressing cholera and surgical pieces. 8- Cancer cells themselves, cell membrane components separated from the returned cells according to conventional methods, and further separated from the membrane components. 1lI3V"Fue
2nLc6 and/or lt”V”Vn3F u csn
Les(Bioehem, Biophyg, Ram, C
Ommun, +109 mNa11p36-44(1
982)) can be used.

H) There are no particular restrictions on the asthmatic mammals that are immunized with M cells, but
It is preferable to select the cell in consideration of compatibility with the bone marrow lung cells used for cell fusion, and mice, rats, etc. are generally used.

Immunization is carried out by a conventional method, and the above-mentioned immunogen is appropriately diluted in physiological saline, phosphate buffered saline (PBS), etc., and then administered to the animal by intravenous or intraperitoneal injection. . More specifically, for example, purified l'V3Fuc2nLe6 and/or 13V3■"Fn
When using a3nLeg, dilute it with PBS% to an appropriate concentration, and add Salmonella minnesota (Salmonella minnesota).
lamino m OtlL) and bovine blood antialbumin (BSA) were administered to the animals several times every 2 to 14 days to give a total dose of 1 to 100 μf/mouse as glycolipid. It is preferable to do so. When membrane components or cells themselves are used as immunogens, the procedure is similar; for example, when membrane components are used, the total dose is about 1 to 100 η/mouse, and when cells themselves are used, the total dose is about I O'~l O' pieces/
It is preferable to make it about the same level as Maunu.

As the immune cells, it is preferable to use spleen cells extracted about 3 days after the final immunization.

The immune cells thus obtained are then fused with bone marrow lung cells. Bone marrow lung cells include known clusters of cells, such as N5-1 in mice. P3, P3-Ul, X45
, SF3, X63.6°5.3, Y3. in rats.
All, 2.3, etc. are used. The fusion reaction is carried out according to a known cell fusion method, for example, by incubation in a medium in the presence of a fusion promoter.

As a fusion accelerator, for example, ethylene glycol (
PkG) and Sendai virus (HVJ) were used.
Furthermore, if desired, an adjuvant such as dimethyl sulfoxide can be added to the mixture by cooling to increase the fusion efficiency. The ratio of immune cells and bone marrow lung cells used is the same as in the general method, 1
1- For example, it is sufficient to use about 1 to 10 times as many immune cells as one myeloma specimen. As the medium for the above fusion, for example, MEM medium used for the proliferation of myeloma cell lines, Dulbecco's modified medium, RPMI-1640 medium, and various other usual media used for this type of cell culture. It is usually best to remove serum supplements such as Fe2.

For fusion, a predetermined amount of the above immune cells and bone marrow lung cells are thoroughly mixed in the above medium, centrifuged, the supernatant is removed, and a PiG solution preheated to about 37°C, for example, an average molecular weight of 1,000 to
This is carried out by adding about 6,000 to a normal medium at a concentration of about 30 to 60 w/ma%.

Thereafter, the procedure of sequentially adding an appropriate medium, centrifuging, and removing the upper 12-ml solution is repeated to form a high cyridoma.

Isolation of the desired hybridoma is carried out by culturing the cells after the cell fusion described above in a normal hybridoma selection medium. The myeloma cell line described above has hexanthin guanine phosphoribosyltransferase (HGP).
RT)-deficient strains, therefore, cannot grow in AT medium (a medium containing hypoxanthine, aminozothelin, and thymidine).Therefore, it is best to select cells that grow in HAT medium.

The cells can be cultured in the HAT medium for a sufficient period of time, usually from several days to several weeks, to kill cells other than the target hybridoma (unfused cells, etc.).

The hybridoma thus obtained can be searched for a strain producing the antibody of interest and cloned into a single clone according to the usual limiting dilution method.

The search for the producing strain can be performed, for example, by the ELISA method [Engva
ll, E, ; M@th, Enzymol, 70
# 419-439 (1980): l and plaque method,
Nutto method, agglutination reaction method, Ouehterlong method,
It is carried out by various methods generally used for antibody detection such as RIA method [[Hyperidoma method and monoclonal antibodies], published by R&D Planning Co., Ltd., 1932.
March 5th, 2017]. More specifically, a table made of cloth “Fue”
nLc4, l”V”Fue2nLe6 or 1i13V3
■Using a plate coated with Fuc3nLeg, for example, cholesterol and lecithin, this is reacted with the culture supernatant of the test hybridoma, and the presence of antibodies that bind to the purified glycolipids is determined according to a conventional method, for example, Anti-mouse immunoglobulin (when using fusion trap mice) Next, confirm the extraction by reacting with 125 knee protein A (Sigma), and select the desired antibody-producing strain. (J,
B, C6, 257, 14865-14874 (1982
); Vlvology, 93, 111-126 (19
79):].

The thus obtained hybridoma producing the monoclonal antibody of the present invention can be subcultured in a conventional medium and can be easily stored for a long period of time in liquid nitrogen. As a representative of this hyperdoma, the present inventor maintains one obtained in the Examples described later in a state where it can be divided by itself.

15- Monoclonal antibodies Y8a, YSd of the present invention from the specific hybridoma obtained as described above.

YSe and YSf can be obtained by culturing hybridomas in tissue according to conventional methods and separating them from the culture supernatant, or by administering hybridomas to compatible mammals, allowing them to grow, and separating them from their ascites fluid. method etc. will be adopted.

The former method is good for obtaining high purity products, and the latter method is excellent for mass production.

The monoclonal antibody of the present invention thus obtained is
Since it selectively reacts with specific 7-cosyl lactosamine-type glycolipids derived from cells, it is extremely useful for the diagnosis, treatment, and research of humans. , Next, the explanation will be given with examples and test examples.

16- All cultured cancers and cell lines used in Examples and Test Examples are known and described in the following literature.

(1) Non-malignant cell line WI-38a Exp, Ca1l Rss, 25.
585 (1961) L-5: Nippon Antibody Research Institute Co., Ltd. Crow, F ditto Prent, B ditto Crow, B ditto Kasn@r, B ditto (21 Acute Cell MKNI: Lancets3.July+7~ l
O (1982) Ca-ncer R611, C42#
60 1-608 (1982) MKN2 Same as above MKN28 Same as above MKN45 Same as above MKN74 Same as above PCI: Kame Shin Igaku, Dango, 1861 (1973) PO2
: Cancer Res, 42, 601-608 (
1982) PO7 same as above PO2 same as above pc101 Machigami QG 56 same as above QG90 same as above RT4: Immunoeh@m1stry+ 15
+ 429-436 (1978) J, Natl, Ca
nc@r In@t, Re*209-214 (1977
) HeLaN Yo5KOV3 Same as above 2102: Cancer Res, 40,796-80
2 (1980) KGI: Blood, 61/1.171-179 (1
983) HL60: Eur, J, Imm, 13.3
06-312 (1983) K562: lip, C@ll Re1, 146/2
*428-435 (1983) THPI: Int, J, Cancers 26 s
171-176 (1980) LXI: Canesr, 42.2269-2281
(1978) 11J23: Proc, Nat, Aea
d, 5cion 79 s 3637-3640 (19
82) TG115: Patient's liver cancer tissue was used.

Example 1 19-fPih glycolipid, 13V”Fue2nLe
6 [Bioehem, BiophyS, Ram, Co
mmun*+ 109 mp 36-44 (1982
)) A 50 μt ethanol solution containing 20 μf was mixed with 800 μl of phosphate buffered saline (pH 4,0). Historically, this solution was treated with Salmonella minnesota [Eu
r, J. Bioehem, 124 e p 116-1
22 (1971)) was mixed with 250 μt of suspended phosphate buffered saline. Mixing was performed at 40°C. Next, a portion of the resulting suspension containing 5 μV of the glycolipid is taken and administered intravenously to Balb/c mice. From then on, the same amount of Glycolipid 2μ was administered 3 times every 4 days. Three days after the final immunization, the spleens are removed and the spleen cells are washed three times with RPMI-1640 medium. Mouse myeloma cell line sp2 [Clinical Immunology 13, No. 11, P912-919 (1981):
] After washing in the same manner, 1XIO' of SF3 and 4X1O' of the above tile cells were placed in a 50-centrifuge tube and mixed. 200x? After centrifugation for 5 minutes, remove the supernatant with a /e stool pipette. It was kept warm at 37℃? Liethylene glycol 1500 (Bastman, Ina, ) 50
RPMI-164g solution 1- w/ma% was added dropwise,
Mix slowly for 2 minutes. 15%FC kept at 37℃
Add 8.1 mM pyruvate of RPMI-1640 (hereinafter referred to as complete RPMI-1640J) IWLl for 1 minute, then add the same amount of complete RPMI-1640 for 1 minute, then dropwise add 8-1 complete RPMI and incubate for 2 minutes. Stir slowly.

After centrifugation at 200Xf for 5 minutes, remove the supernatant, suspend in complete RPMI-1640 kept at 37°C to a density of 1 x 10' cells/-, and inoculate 100 μt each onto Microtenu-II plate (Falcon). Then, culture in a 5% carbon dioxide incubator at 37°C. 1.0X after 24 hours
l O-'M Hyboxanthin, 4. OX 10-7
Add 100 μt of the above complete RPMI-1640 (hereinafter referred to as "HAT medium") containing M aminopterin, 1.6 X l 0-5 M temizone to each well. Thereafter, half of the supernatant was replaced with fresh HAT medium on days 2.3.5.8 and 11, respectively, and on day 14, half of the supernatant was replaced with 1.0×10'M HAT medium. Xanthine, 1.6 x 10
-'M Change to complete RPMI-1640 (hereinafter referred to as "IHT medium") containing thymizone. Similarly, No. 18.22
．． On days 25 and 26, half of the supernatant was replaced with waste medium, and the second
On the 8th, half of the supernatant is replaced with complete RPMI-1640. Thereafter, the cells are grown and maintained using this complete RPMI-1640.

The hybridoma thus obtained was cloned by the limiting dilution method. That is, hyperidoma 2.
Complete RPMI-16 to give 5XlO cells/d, Ba1b/c Y mouse thymocytes 4X1 gll/ml.
40 and cultured in a 200-well plate at 5 hybridomas/well. The proliferating hyperdomas are further transformed into hybridoma 0.0 in the same manner.
It was cloned as 25 cells/well. To search for a clone producing the desired antibody, use purified 23-13FuenLe4, 1Qnf/well of l"V'Fue2nLe@ or Bar3V3■"Fue3nLeg, colenutrol 30n, f/well, and lecithin 50nf/well.
Using a well-coated 96-well plate (Dynatic Laboratories), extraction was carried out by the in-phase method using rabbit anti-mouse immunoglobulin (Cabbell) and 125-nitrogen protein (Sigma). The desired hybridoma represented by clone mysa was thus obtained.

Example 2 ■ 1.5 f of tumor tissue or tumor cells (TG l l 5 lung tumor cells or FiMKN74 cells) was added to 10 kg distilled water [aprotinin (zolotease inhibitor; Sigma Chemical Co., Ltd. #) J 10 kg international unit Add to the mixture and homogenize with a Doune 24-homogenizer. In an ice-water bath approx.
After 0 stokes, the homogenized mixture was heated to 200
Centrifuge at 0 rpm for 10 minutes, separate the supernatant, and then centrifuge for 35 minutes.
．． Centrifugation was performed for 1 hour at 00 Orpm. The obtained pellet was suspended in lO- of distilled water containing aprotinin, and the protein concentration was adjusted to 2.5/-.

■ 0.5-(
Contains 1.25 M of protein) B a 1b / 1
! Administer intraperitoneally to mice. Administration was carried out every 4 days, a total of 4 times, and the spleen was removed 3 days after the final administration.

■ The spleen was removed on the 3rd day after the single administration of the TG115 component in (■) above.

(2) Using 0.5 m of the MKN 74-layer component obtained in (2) above (containing 1.25 mm of zolothiin), immunization was performed by intravenous administration using the same schedule as in (2) above to obtain splenic cells.

(2) Using the tile cells obtained in C) to (3) above, the desired hybridoma was obtained in the same manner as in Example 1 above. The representative clone was cloned Nu ysa (TG1i5 membrane Jl
ill, selected from the 4-time immunization group), YBt (
TG 115 (selected from the M component single immunization group) and ysf (selected from the MIGJ 74 membrane component immunization group).

Example 3 Clone Nn Y3a obtained in Examples 1 and 2.

YSd, YSe, and YSf hybridomas were cultured in complete RPMI-1640 medium in a 5% carbon dioxide incubator at 37°C for 48 hours. The culture medium was centrifuged (3,000 rpm, 10 minutes) to obtain a culture supernatant containing the monoclonal antibody of the present invention (indicated sequentially as YSa XYSd, YSe, and YSf).

(2) Clone ImYSa obtained in Examples 1 and 2.

Each hybridoma of YSd, YS・and YSf l×10
6 were suspended in RPMI-1640 medium 0.5-
a1b/e mice were administered i.p. After 2 to 3 weeks, the accumulated ascites was collected and treated with antibodies Y8c and YSd, respectively.
, ascitic fluid containing YBt and YSf was administered to 2-5-/mice. The incidence of these antibodies was 0.2 to 1/-.

・Test ψ'1 (1) Immunoglobulin class: 27-rabbit antibodies against various mouse immunoglobulin classes (Litton, Eion@tieo, In
c. Kenmington.

MD20795) and 12J-labeled protein protein according to the method of Yeh et al. [Ming-Ya
ng Ysh at al, , Proe, Natl.
Acad.

Sei, USA, Vol, 76, Nn6,, p
p, 29.27-2931.

(1979:), ).

The results are shown in Section 1-i below.

(2) Antibodies Y8a, Ysa -, YBt, and YBt against various glycolipid antigens: ■ As ha antigen, 1"F'acnL; 4, 1%"
Fu, e2. nLc6, 28- [3■! ■”, FuclnLe4 > ■”FuenL
e6 or 13FuenLc6c, Bioeh*m, Bi
ophys, ROlCommun, + 113 *p7
91-798 (1983)), its 1Onf/
The antibody of the present invention obtained in Example 3 (1) (and its serial dilution) was added to cholesterol (30nf/well) and helecithin (50nf/'yel) (Tecordontavinylstriff (Konu) Co., Ltd.). ) was reacted.

After thorough washing with PB8, the amount of antibody bound to the purified antigen was measured using rabbit anti-mouse immunoglobulin as a second antibody and then 115-protein antibody 1, and the binding rate was determined by radiation counting.

The results for antibodies YSa, YSd and YSe17 are shown in Figures 1.2 and 31, respectively. In the figure, the vertical axis is the radiation count number (CPM) of the bound 1261-protein
X 1O-3), and the horizontal axis shows the dilution factor of the antibody. In addition, each symbol indicates the following.

・−・; ll”V”Fue2nLe6△−△; ■３
FuenLe6 0-〇: l3FucnLe4 0H ko;111■3■”Fue3nLeBmom: l
3FuanLc6 ■ Initial 30nf/well of purified antigen, cholesterol (90nf/well) and helecithin (1
The reactivity with the antibody of the present invention was examined in the same manner as in (2) above using vinyl strips coated with 50nf/well) and vinyl strips in which the antigen concentration was serially diluted.

Each antibody was prepared by adding 100% of the antibody obtained in Example 3 (1) above.
It was diluted twice and used. Antibody Y8a, Y8d%ys
- and YSf results are shown in Figures 4.5.6 and 7, respectively. In each figure, the vertical axis is the combined 12+11
- The number of emission counts of protein A is shown, and the horizontal axis shows the dilution times relative to the initial concentration of the antigen. Also, the meanings of the symbols in each figure are the same as above, and X-X in Figure 7 is ■"Fu
Indicates cnLeH.

As is clear from FIGS. 1 to 7, the antibody of the present invention Y8a%
Y8d XY8@ and YSf are Otori “%l”FuelnL
It has reactivity to c6 and/or 13V8■''Fuc3nLcg, which is clearly distinguished from reactivity to l''FuenLe4. The recognition site of the antibody of the present invention estimated from the above test results is shown in FIG.

(3) Reactivity with each young human cell: 31- The cells fixed on the plate were treated with phosphate buffered saline (pH 7.4) containing 5% bovine serum albumin for 1 hour, and then treated with the antibody YSA (from the above example). 3(2) antibody obtained in 10
0 times dilution) was added and incubated for 18 hours. Furthermore, the cells on the plate were treated with a 1000-fold dilution of a rabbit antibody that reacts with mouse IfG3 as a second antibody, and then 121S1-labeled.

Treated with protein A. In addition, antj-881ii
Mu-1 was used as a comparative example. However, 1. In this case, the second antibody used was a 1000-fold dilution of a rabbit antibody that reacts with mouse ety.

The results are shown in Figure 9. In Figure 9, the horizontal axis is MKN 74
1 that was combined when using ! ! The binding rate is shown as 32%, where the radioactivity of labeled protein A is 100%.

As shown in FIG. 9, antl-5SEA-1 reacts with various cells and also reacts with non-malignant B cell lines, whereas the antibody YSa Fi of the present invention reacts with specific cancer cells, such as the cancerous cell line MKN74. , lung cancer cell line pc7
and selectively reacts specifically with the monocytic leukemia cell line THP-1.

[Brief explanation of the drawing]

Figures 1, 2 and 3 show the antibody YBa XY of the present invention, respectively.
Drawings showing the reactivity of Sd and YSe to various glycolipid antigens at various antibody concentrations, 4th
．． Figures 5.6 and 7 show the antibody Y8aXys of the present invention, respectively.
Figure 8 shows the reactivity of aSys and YSf to various glycolipid antigens when the antigen concentration was changed.
A drawing showing the presumed recognition site of Sf, FIG. 9 shows the antibody Y of the present invention.
FIG. 8 is a drawing showing the reactivity of 8d with various human cells. Applicant Otsuka Pharmaceutical Co., Ltd. 35- To dimension o < To D --- , -01X 8-〇 separate coeo < D To, -01 To ψ, 0IXl/lJd:) 0 CD, -01X people d

Claims (1)

[Claims] 1. General formula % [In the formula, Gat is galactose, GteNAC is N-acetylglucosamine, F is 7 course, Gtc is glucose, C@r is ceramide, and n is l or 2. sifcosyl lactosylceramide, a fucosyllactosamine glycolipid derived from human cancer cells, represented by
-norh*xaomyleeramide) and/
or trifucosyl neolacto-n
It is generated from a hybridoma obtained by fusion of myeloma cells with the immune cells of a sentinel mammal immunized with oroctao-sylceramld. G1
．． e SC@r and n have the same meanings as above] Reacts with one or two types of 7-cosyl lactosamine disc glycolipids derived from human cancer cells represented by (2) the following formula GiL191→4GteNAC#l- +3Gatβl-
+4G4cβl-+lC*r3 ↑ Fueα1 [In the formula, Gat, GteNAC, Fue 5Gla and Car have the same meanings as above] Lacto-fucopentaosyl (1) ceramide coated with
A monoclonal antibody characterized by not reacting with cerarnid.).