JPH0661264B2 - Monoclonal antibody-producing hybridoma for drug-resistant cancer - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hybridoma which produces a monoclonal antibody against a drug resistant cancer cell.

It has been a serious problem that a cancer cell having resistance to an anti-cancer drug is selected and appears when the cancer is treated by chemotherapy. In order to overcome this resistance, it may be possible to increase the dose of the anticancer drug. However, increased doses unnecessarily afflict patients with the side effect of damaging normal cells.
It would be conceivable to use other types of anti-cancer agents as another means of overcoming resistance. However, in this case, cancer cells that have once become resistant to one drug are often referred to as so-called “pleiotropic drug resistance”.
The effect is often small.

Therefore, in order to overcome such drug resistance of cancer cells, it is an important subject to establish an effective drug or method for cancer cells having few side effects, high selectivity and multidrug cross-resistance. Has become.

[0004]

2. Description of the Related Art Monoclonal antibodies having selectivity for cancer cells showing multidrug cross-resistance have already been prepared [J. Clin. Onc.
ology), Vol. 3, pp. 311 to 315 (1985)
Year)〕. This is a monoclonal antibody that reacts with a glycoprotein having a molecular weight of 170,000 to 180,000 daltons that specifically appears on the cell membrane of a cancer cell exhibiting multidrug cross-resistance. But,
The resistant cell line used in preparing this monoclonal antibody was derived from Chinese hamsters rather than humans, and there is no report on the sensitivity of resistant cancer cells to drugs by this monoclonal antibody.

It is therefore understood that this prior art monoclonal antibody could not be used for the selective treatment of human drug resistant cancer cells.

[0006]

The problem to be solved by the present invention is to overcome the drug resistance of cancer cells, and thus to treat cancer cells with few side effects, high selectivity and multiple drug alternate resistance. To provide effective drugs.

[0007]

[Means for Solving the Problems]

<Summary> The present inventors have found that a monoclonal antibody produced by a hybridoma obtained by fusing mouse splenocytes immunized with adriamycin-resistant tumor cells and mouse myeloma cells shows multidrug cross-resistant cancer cells. The present invention has been completed by finding that it selectively inhibits growth or enhances sensitivity to the drug.

That is, the monoclonal antibody-producing hybridoma according to the present invention is a human myeloid leukemia cell line K.
It was prepared by cell fusion of splenocytes obtained from mice immunized with 562 adriamycin resistant strain K562 / ADM and mouse myeloma cells. However, this monoclonal antibody has the following (a) to (d)
Is defined by

(A) Human myeloid leukemia cell line K562
Produced by hybridizing splenocytes obtained from a mouse immunized with the adriamycin-resistant strain K562 / ADM of E. coli with myeloma cells of the mouse. (B) Reacts with an adriamycin resistant strain but does not substantially react with an adriamycin sensitive strain. (C) The ability to inhibit the cell growth of an adriamycin resistant strain or to enhance the sensitivity of the cells to vincristine or actinomycin D. (D) Being of the IgG isotype.

<Effect> As described above and as is clear from the experimental examples described below, whether the monoclonal antibody produced by the hybridoma according to the present invention selectively inhibits the growth of cancer cells exhibiting multidrug cross-resistance, Alternatively, it has the ability to increase the sensitivity to the drug.

Therefore, the monoclonal antibody produced by the hybridoma according to the present invention is one of the important objects of establishing a drug or method effective against cancer cells showing few side effects, high selectivity and multidrug cross-resistance. It can be one solution.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a monoclonal antibody-producing hybridoma. This hybridoma will be described below together with the monoclonal antibody that is the product thereof.

[0013] Since several reviews and publications have already been known regarding the production of monoclonal antibodies, including the cell fusion method, regarding the necessary information other than the following explanation of one embodiment of the present invention, those documents are referred to. Please refer to.
Some of the relevant references include, for example, Garfre and Milstein: "Methods in Enzymology", G. Galfre, C. Milstein: Methods Engymol., 73, 3-46 (1981). ) And Goding: “Monoclonal Antibodies: Principles and Practices” (Academic Press, 1983) (JWGoding: Monoclonal Antibodies: Princ
iples and Practice, Academic Press, 1983).

Production of Hybridoma / Monoclonal Antibody (1) Selection and Establishment of Adriamycin-Resistant Cancer Cells By the present inventors, a human myeloid leukemia cell line (K562 / VCR) resistant to vincristine, which is an anticancer agent for vinca alkaloids, has already been prepared. ) Has been established, but
This resistant strain already shows mild cross-resistance to adriamycin, which is an anthracycline anti-cancer drug ["Gann"] Vol. 74, 751-75.
8 (1983)]. In a specific embodiment of the invention, this strain is used as the starting cell line. This strain is a cancer research group, cancer chemotherapy center (1-37 Kamiikebukuro, Toshima-ku, Tokyo)
It can be freely obtained from -1).

First, the vincristine-resistant cell line K562
/ VCR containing RPMI 1640 (10% fetal calf serum (FC
S)) is cultivated in S)), and the concentration of adriamycin in the grown cells is sequentially increased at a ratio of about 3 times, thereby selecting a high-concentration drug-resistant strain. Obtain a stable cell line that does not lose its resistance even when the selected drug-resistant strain is grown for about 1 year in the presence of the highest concentration of drug that the resistant strain can grow and then cultured in a drug-free medium. .

(2) Preparation of immunized animal splenocytes The obtained adriamycin-resistant human myeloid leukemia cell line K
562 / ADM was washed once with 0.5 ml of Hanks' balanced buffer saline (hereinafter referred to as HBBS) so that 10 ⁷ cells were obtained per mouse, and then suspended in the same amount of HBBS, Female Balb / c 4-6 weeks old
The mice are intraperitoneally administered. Similarly, the administration is continued once a week until the antibody titer sufficiently rises, and 3 days before cell fusion, 10 ⁶ cells are suspended in 0.1 ml and intravenously administered as a booster. The spleen is aseptically collected from the immunized animal thus obtained. Aseptically loosen the removed spleen using tweezers on a petri dish, take a portion of it,
The number of cells obtained is calculated.

In order to measure the antibody titer, the antibody titer of the anti-adriamycin resistant strain is examined by the enzyme immunoassay by the solid phase method shown below. Plate Pretreatment: Falcon Plate 391
50 μl of a poly-L-lysine 0.001% solution is added to 2 per well, reacted at room temperature for 30 minutes, drained of water and air-dried.

Binding of cells to the plate: K562
The parent strain and the K562 / ADM resistant strain were suspended at a concentration of 2 million cells / ml each, and 50 μl (well, 100,000 cells) per well was prepared and dispensed into each well. Bind to wells by centrifugation at 1,000 rpm for 5 minutes. The binding is then made more complete by adding 50 μl of 0.5% glutaraldehyde per well.

Blocking: To mask excess binding groups, 200 μl per well of bovine serum albumin (BSA) dissolved in RPMI1640 at 3% is added and treated at room temperature for 30 minutes.

Addition of measurement sample: 50 μl of the sample for measuring antibody titer was added to each well of K562 and K562 / ADM for 50 μl per well, and reacted at room temperature or 37 ° C. for 2 hours. . Then phosphate buffered saline (PBS)
, Wash 4 times.

Addition of secondary antibody: Goat peroxidase conjugated-anti-mouse Ig antibody F (a
b ') ₂ fragment (manufactured by US Cappel)
The solution diluted with 1500 times PBS is added to each well and further reacted at room temperature for 2 hours.

Determination: Ortho-phenylenediamine as a substrate for peroxidase was added to each well, the reaction was stopped with sulfuric acid, and then the presence or absence of color development between K562 and K562 / ADM is determined by the naked eye or an auto reader.

(3) Preparation of mouse myeloma cells Examples of bone marrow cell lines include mouse-derived 8-azaguanine-resistant myeloma cell line P3.X63.Ag8.65.
3 [Journal of Imm
unology) 123, 1548-1550 (19)
79)] is used. On the day of fusion, the number of cells should be 2 × 10 ⁷ or more. This strain is CRL-15 in the American Type Culture Collection in Maryland, USA.
It is registered as 80
Laboratory Incorporated (Flow Laborator
y Inc.).

(4) Cell fusion The splenocytes obtained from the animal immunized with (2) and the myeloma cells obtained in (3) have splenocytes: myeloma cells = 7:
Mix to 1 and polyethylene glycol 4000
Cells are fused in RPMI-1640 medium containing 43% and 13% dimethyl sulfoxide, respectively.

The fused cells were mixed with RPMI-164 containing hypoxanthine, aminopterin and thymidine (hereinafter abbreviated as HAT) in a 96-well plastic plate.
Grow for 7 days in 0 medium and in medium without HAT. During this period, the medium is replaced every 3 to 5 days.

Approximately 2 weeks after the fusion, the surviving cells were examined for the presence of antibodies selectively binding to K562 / ADM in the culture supernatant by the enzyme immunoassay method described in (2) above. To do. For positive wells, positive cells are cloned by repeating the limiting dilution method using RPMI-1640 containing 20% fetal bovine serum as a diluent.

(5) Preparation of Monoclonal Antibody Pristane was pretreated by intraperitoneally administering 0.5 ml per mouse to the mice, and 10 ⁷ hybridoma cells producing the desired antibody were intraperitoneally administered to each mouse. Administer. Since the ascitic cancer of the hybridoma becomes ascites cancer about 2 weeks after the administration, the accumulated ascites is collected, and the presence or absence of accumulated antibody is examined by the enzyme immunoassay method (2).

When ascites is stored, the supernatant after centrifugation is divided into aliquots and stored frozen at -70 ° C.

Ascites fluid was added to the antibody for further purification.
Salting-out is performed at 4 ° C. with% saturated ammonium sulfate, and gel filtration is performed using Sephacryl-400 (manufactured by Pharmacia, Sweden). The protein is quantified by the Lowry method.

Properties of Monoclonal Antibody The class of the monoclonal antibody obtained as described above can be determined by using an antibody of each class of rabbit anti-mouse Ig (manufactured by Kappel Co., USA). Further, the localization of the antigen in the cells can be known by observing the fluorescence under a microscope using a goat anti-mouse Ig antibody (manufactured by Kappel, USA) to which FITC which is a fluorescent chromophore is bound.

The selectivity of the drug-resistant strain for K562 / ADM was determined by the enzyme immunoassay of (2) using the parent strain K562 as a control, and the degree of inhibition of cell proliferation was measured for each cell line. You can look it up.
Further, the change in the sensitivity of the drug-resistant strain to the drug due to the monoclonal antibody can also be examined by measuring the degree of cell growth inhibition by coexisting with the monoclonal antibody in the presence of various concentrations of the drug.

Specific examples of the monoclonal antibody according to the present invention include those having an isotype of IgG ₃ , IgG _2a , IgG ₁ and the like. In the present invention, these are designated as MRK4, MRK16 and M, respectively.
It is named RK17. Typical of these are MRK16 and MRK17. Monoclonal antibodies MRK16 and MRK17 have selective growth inhibitory action or drug susceptibility increasing action on drug resistant human cancer cells (see Example II).

[0033]

Example I Preparation of monoclonal antibody a) Selection and establishment of adriamycin resistant cell line For selection of adriamycin resistant cell line K562 / ADM, vincristine resistance prepared from the same parental human myeloid leukemia cell line K562 Cell line K562 /
VCR was the starting cell line.

First, K562 / VCR was added to RPMI-1640 containing 15 nM adriamycin, which is the IC50 of this cell line (concentration that inhibits cell growth by 50%).
After culturing in a medium (containing 10% fetal bovine serum) for 1 week, growing cells were selected. Next, the drug concentration of adriamycin was increased about 50 times to 50 nM, and for the resistant cells obtained after culturing for 1 week, the amount was further increased to 150 times.
The drug concentration is gradually increased with nM, and finally 45
It was possible to obtain adriamycin resistant cells that could grow even in the presence of 0 nM adriamycin.

The adriamycin-resistant cells were stabilized by continuing to culture the drug in a medium containing 500 nM for about 1 year, so that the resistance was not lost even when cultured in a medium containing no adriamycin for about 3 months thereafter. It became a resistant strain. The present inventors have set this as K562 / ADM.
I named it.

B) Immunization of mouse and cell fusion With K562 / ADM obtained in a), an amount of 10 ⁷ cells per mouse was suspended in 0.5 ml of HBBS, washed once, and then 4 to It was intraperitoneally administered to 6-week-old female Balb / c mice. In the same manner, the administration was repeated once a week for 6 weeks continuously, and 10 ⁶ cells were added to 0.1 ml of HB as a booster in the last week.
The suspension was suspended in SS and intravenously administered, and three days later, the spleen was aseptically taken out. Spleen cells were obtained in an amount of 1.4 to 3 × 10 ⁸ per mouse. The spleen was loosened with tweezers to give a suspension.

Cell fusion was performed according to the method of Köhler and Milstin. That is, 1.4 × 10 ^{8 of} these spleen cells were treated with RP containing 43% polyethylene glycol 4000 and 13% dimethyl sulfoxide.
2 × 10 ⁷ P3.63 · A in MI1640 medium
Fused with g8653 myeloma cells.

C) Selection of hybridomas After fusion of the cells, they were grown on 96-well plastic plates in HAT medium for 7 days and in HAT-free medium for 7 days. 300 ~ per mouse
400 wells were cultured, and about 75% of the wells showed cell growth. The culture supernatants of these wells were analyzed by enzyme immunoassay for K562 and K562.
The reaction to 562 / ADM was about two-thirds.
Equally positive color development was observed for both K562 and K562 / ADM in the wells, and both were unreactive in the remaining third well. Furthermore, it reacts with 0 to 3 K562 and K562 / ADM per 300 to 400 wells derived from one mouse, but apparently K562 / A.
Wells that responded strongly to DM were found.

Approximately 6000 using a total of 18 mice
The wells were screened and K562 <
Twenty-five wells with K562 / ADM reactivity were found.

D) Cloning of hybridoma Reaction-positive cells were cloned from the 25 wells selected in c) by the limiting dilution method. RPMI-16 containing 20% fetal bovine serum as the diluent
40 was used to dilute the cells so that 0.5 to 5 cells were distributed per well, and the cells were cultured. For each well, the presence or absence of the antibody in the culture supernatant and the property of the well were examined by enzyme immunoassay.
Furthermore, repeating cloning by the limiting dilution method,
It was possible to obtain 25 stable hybridoma clones.

E) Production and purification of antibody 16 clones out of 25 hybridomas obtained in d) were intraperitoneally administered with 10 ⁷ mice per mouse to develop ascites cancer. After 2 weeks, 5-10 ml of ascites was obtained per animal. The remaining 9 clones were stored frozen (at -70 ° C). The total amount of protein in the ascites was 5 to 15 mg / ml. 45 of these ascites
After salting out with% saturated ammonium sulfate, the antibody was purified by gel filtration with Sephacryl-400 (Pharmacia, Sweden).

Example II Monoclonal Antibody MRK-1
6 and Properties of MRK-17 a) Antibody isotype The 16 types of the obtained monoclonal antibodies were determined for their isotypes using a kit using antibodies of rabbit anti-mouse Ig each isotype manufactured by Kappel, USA. As a result, IgG ₃ is one (MRK4), _{IgG 2a} which one (MRK16) and IgG ₁ is 1 (MRK17) were obtained, it revealed that all the other belongs to IgM isotype.

B) Determination of Location of Antigen by Fluorescent Antibody Method Using FITC-conjugated goat anti-mouse Ig antibody (Kappel), the binding sites between the monoclonal antibodies of MRK16 and MRK17 and the antigen on K562 / ADM cells were determined. As a result of examination, in both cases, ring-shaped fluorescence was observed on the cell membrane surface. That is, all the antibodies recognized the antigen localized in the membrane of K562 / ADM cells. As a control, even when the parent strain K562 was treated in the same manner, no such fluorescent color development was observed.
It was revealed that both 16 and MRK17 have strong selectivity for K562 / ADM. In addition, MRK16 and MRK17 showed K5 by radioisotope immunoassay.
It does not react with 62 cells and is a resistant strain K562 / ADM
It became clear that it reacts with a very high selectivity (see FIG. 1). That is, 10 ⁶ cells were reacted with ascites dilutions of MRK16 and 17 respectively, and the cells were washed and then ¹²⁵ I-labeled sheep anti-mouse Ig F (ab ′) ₂ fragment (Amersham, England) was used.
Reacted with cells at 4 ° C for 30 minutes, washed and reacted with cells M
The results of measuring the reactivity (cpm) of RK16 and RK16 were as shown in FIG.

C) Identification of Other Drug-Resistant Strains Adriamycin-resistant strain 278 of human ovarian carcinoma cell line 2780, which has been the only human tumor cell line to date, is considered to be few in number, so far.
Regarding 0 ^AD , the reactivity with MRK16 and MRK17 was examined by using a radioisotope immunoassay, and both monoclonal antibodies reacted strongly with 2780 ^AD of the resistant strain as compared with 2780 of the parent strain (see FIG. 2). ). That is, 3 × 10 ⁵ cells were seeded on a dish, and 24 hours after the cells adhered to the dish, the reactivity (cpm) of MRK16 and 17 was the same as that shown in FIG.
The result of measurement was as shown in FIG.

D) Selective Cell Proliferation Inhibition of MRK17 Against K562 / ADM Monoclonal monoclonal antibodies were used for the resistant strain K562 / ADM and its parent strain K562, and the resistant strain 2780 ^AD and its parent strain 2780 belonging to different cell lines for comparison. Ascites containing the antibody MRK17 was serially diluted to examine its inhibitory effect on cell proliferation (see FIG. 3). That is,
2 × 10 ⁴ K562 and K562 / ADM cells,
And 6 × 10 ⁴ 2780 and 2780 ^AD cells were incubated with ascites dilution of MRK17, 7
The results obtained by measuring the number of cells after 2 hours and calculating the percentage of growth inhibition were as shown in FIG.

As a result, almost no inhibition was observed for the respective sensitive parent strains K562 or 2780 even when the antibody concentration was increased up to a 1/400 dilution, whereas the drug-resistant strains 2780 were inhibited. Approximately 30% growth inhibition was observed in ^AD at the same antibody concentration, and K562 / ADM
Up to 80% growth inhibition was observed, and with 1 / 100th dilution antibody, 90% or more growth inhibition was observed (triplicate data SD <4%).

When a similar experiment was carried out using MRK16, a slight growth inhibition of up to about 12% was observed with respect to K562 / ADM. That is, it can be said that a major feature of MRK17 is that it has a strong growth inhibitory action selective for drug resistant strains.

E) K562 / ADM according to MRK16
Effect of MRK16 on drug sensitivity in d) above, drug resistant strain K562 / ADM
Although the direct inhibitory effect on the growth of M. pylori was mild, vincristine was added at a concentration of 0.5 μg / ml to this experimental rate, and the growth inhibition of resistant strains of up to about 75% was observed in the presence of MRK16. (See FIG. 4). That is, 2 × 10 ⁴ K562 / ADM cells were incubated with an ascites dilution of MRK16 in the presence and absence of 500 ng / ml of vincristine (VCR), 72
The results obtained by measuring the percentage of growth inhibition after time were as shown in FIG.

Using Vincristine, K562 / VC
The effect of MRK16 on the accumulation in R cells was found to be increased by about 60 to 70% as compared with the control experiment in which no antibody was added (see FIG. 5). That is, 2 × 10 ⁵ K562 / VCR cells were subjected to [ ³ H] VCR (10) in the presence and absence of MRK16 (ascites 1: 200 dilution).
(0 nM) and the amount of vincristine taken up by the cells was quantified. The results are shown in FIG.

That is, MRK16 has a possibility of recognizing the point of action involved in pumping out intracellular drug, which is enhanced in resistant strains, to the outside of the cell. here,
Using 2780 ^AD of a resistant strain other than K562 / ADM,
When the change in sensitivity to vincristine was observed, the IC50 value was decreased by 20 to 30%, that is, the sensitivity to the drug was increased.

In addition to vincristine, MRK16 was also able to enhance the sensitivity of K562 / ADM to actinomycin D by about 3-fold (see FIG. 6). That is, 2 × 10 ⁴ K562 / ADM cells were transferred to MRK16.
The results obtained by incubating at 37 ° C. with various concentrations of actinomycin D in the presence and absence of (ascites 1: 1000 dilution) at 37 ° C. and measuring the percentage of growth inhibition after 72 hours are shown in FIG. It was as shown in 6.

From these results, it can be said that MRK16 is an antibody capable of selectively acting on many anticancer drug resistant strains and enhancing the sensitivity to the drug.

[0053]

INDUSTRIAL APPLICABILITY The monoclonal antibody produced by the hybridoma according to the present invention has the ability to selectively inhibit the growth of cancer cells exhibiting multidrug cross-resistance or enhance the sensitivity to the drug. The thing is
It has been described above in the section “Means for solving the problem”.

[Brief description of drawings]

1 (A) and (B) show the reactivity of MRK16 (A) and MRK17 (B) to human myeloid leukemia cell K562 and its adriamycin resistant strain K562 / ADM cells by radioisotope immunoassay. It is a graph.

2 (A) and (B) are graphs showing the reactivity of MRK16 (A) and MRK17 (B) to human ovarian cancer cell 2780 and its adriamycin resistant strain 2780 ^AD by radioisotope immunoassay. .

FIG. 3 is a graph showing inhibition of cell growth by MRK17.

FIG. 4 is a graph showing cell inhibition by the combined use of MRK16 and vincristine.

FIG. 5 is a graph showing increased uptake of vincristine by MRK16.

FIG. 6 is a graph showing enhancement of the effect of actinomycin D by MRK16.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12N 15/06 C12P 21/08 8214-4B (C12P 21/08 C12R 1:91)

Claims (2)

[Claims] 1. A monoclonal antibody-producing hybridoma prepared by cell fusion of splenocytes obtained from a mouse immunized with an adriamycin resistant strain K562 / ADM of a human myeloid leukemia cell line K562 and mouse myeloma cells. However, this monoclonal antibody is defined by the following (a) to (d). (A) It is produced by a hybridoma prepared by fusing splenocytes obtained from a mouse immunized with an adriamycin-resistant strain K562 / ADM of human myeloid leukemia cell line K562 with mouse myeloma cells. . (B) Reacts with an adriamycin resistant strain but does not substantially react with an adriamycin sensitive strain. (C) The ability to inhibit the cell growth of an adriamycin resistant strain or to enhance the sensitivity of the cells to vincristine or actinomycin D. (D) Being of the IgG isotype. 2. The hybridoma according to claim 1, which is hybridoma MRK16 or hybridoma MRK17.