JP2747774B2 - Cancer foreign body promotion method, cancer foreign body degree determination method, and cancer regression method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for promoting cancer foreignization, a method for judging the degree of cancer foreignization, and a method for regressing cancer using the same. The present invention relates to each of the above methods, which can be performed simply and reliably as one parameter.

[0002]

2. Description of the Related Art Cancer treatment includes surgery, anticancer drugs, radiation,
Various treatments (treatments) such as heat are used. However, the degree of such treatment generally tends to be excessive. For example, in the case of administration of an anticancer drug, the dose is usually administered to a limit amount that the living body can tolerate. Therefore, hematopoietic disorders are often caused by side effects of the anticancer drug, which not only causes a great deal of pain to the patient, but also produces undesirable results in the treatment of cancer itself. In addition, a plurality of treatments are often performed in parallel in cancer treatment, and the degree of side effects increases accordingly. Under these circumstances, there are few side effects,
In addition, there has been a demand for the development of a method capable of exhibiting a therapeutic effect effectively.

[0003] Under these circumstances, immunotherapy for cancer has recently been widely tried clinically.
Current immunotherapy attempts to activate body immunity to immunologically reject cancer cells. Cancer cells are originally
Tumor associated transplantation
antigen: TAA)
Since the antigenicity is extremely weak, the aim is to artificially enhance the antigenicity by modifying the membrane surface of cancer cells with low antigenicity, thereby activating biological immunity and immunologically rejecting cancer cells. Attempts have been made to do so.

[0004] Two approaches are possible to elicit cancer cells that have only weak and unstable TAAs into the host immune response field. The first method is to enhance the weak antigenicity of TAA by exposing the antigen group, imparting a positive charge, or modifying the antigen to enhance the recognition of cancer cells in the host. The second approach does not rely on weak TAA, but instead recruits or induces another highly foreign antigen to the cancer cells, thus rejecting the cancer cells directly by the immune response of the borrowed so-called foreign antigens. It is an attempt to take advantage of it.

When a strong foreign antigen is given to a cancer cell, the living body rejects the cancer cell by targeting the foreign antigen.
In the rejection process, the living body recognizes TAA together with foreign antigens, and after rejection of cancer cells, has a strong antitumor resistance even to parental cancer cells to which no foreign antigen has been given. Giving and introducing a foreign substance antigen to cancer cells is referred to as "transforming cancer into foreign substances".

[0006] Heretofore, as a means for cancer foreignization, a virus ("J. Natl. Cancer Inst.", 42: 413, 1969; "J. Exp.
Med. ", 126: 93, 1967;" Int. J. Cancer ", 21:62, 1978),
Chemicals ("Nature", 271: 463,1978; "J. Exp. Med.", 14
9: 185, 1979), enzymes such as neuraminidase ("Br. J. Ca
ncer ", 23: 141,1969;" Cancer Res. ", 31: 2130,1971),
A chemical variant ("Proc. Natl. Acad. Sci. USA", 7
4: 272,1977; "J. Exp. Med.", 159: 1491, 1984) and the like.

[0007] Among them, the most effective one is foreignization by a virus (antigen). In animal experiments, by artificially infecting a virus that expresses a foreign antigen on the cell membrane surface that is easily recognized as a foreign substance by the host, the antigenicity of cancer cells can be increased, and in normal syngeneic hosts, cancer can be regressed spontaneously. Indicated. The most typical model is when rat cancer cells are artificially infected with a virus susceptible to mice (eg, Friend virus, Gross virus, Lausher virus, etc.). Rat cancer cells infected with such artificial virus infection almost always regress when transplanted into syngeneic or autologous hosts. The xenogenic cancer cells are extremely foreign due to viral infection, and can be regressed by humoral immunity without borrowing from the host cell-mediated immunity. In addition, this regression of xenogenic cancer cells is observed not only for primary cancers but also for metastatic cancers. In other words, an experimental model has been shown for metastatic cancer in which the regression of cancer occurs because of the foreign body created artificially without the treatment of so-called cancer.

[0008] A new cellular antigen (virus-associated antigen: VAA) due to viral infection is formed on the membrane surface of a cancer cell that has been made foreign by a virus. This is recognized and rejected by the rat, which is the host of cancer cells, as foreign. The main cause of the regression of xenogeneic cancer cells is due to the xenobiotic nature of the VAA antigen newly formed on the cell membrane surface, but further involvement in the increase in the antigenicity of existing TAA cannot be ruled out. That is, an increase in immunogenicity.

However, in the case of viral (antigen) foreignization, an appropriate amount of VAA must be expressed in order to induce immunity to TAA, and excessive expression of VAA on the surface of cancer cell membranes may impair immunity to TAA. Instead of inducing, it may decrease ("Cancer Res.", 43: 230
1,1983; "Cancer Immunol. Immunother.", 12: 119, 198
2). In addition, to make the body foreigner, persistent infection of the virus,
In other words, it is necessary to proliferate in a form coexisting with the growth of cancer cells while invading and proliferating in the cells. However, most virus infections are cytolytic and it is difficult to obtain a persistent infection. Moreover, in this case, the virus used is a leukemia virus derived from a mouse, and although its natural infectivity to normal rats is not recognized, its pathogenicity cannot be ruled out. That is, when infectious virus particles are used, their pathogenicity cannot be completely denied.

[0010] Recently, cancer has been made foreign by gene insertion ("Cancer Res.", 47: 3136, 1987). It is necessary to select an appropriate gene for It has also been reported that treatment of cancer cells with a mutagenic substance results in the appearance of clones with high antigenicity.In this case, however, there are problems such as the appearance of clones with high tumorigenicity. is there.

[0011] Furthermore, as an even greater problem, at present, the transformation of cancer cells into foreign matter is merely of biological interest using laboratory animals, and has not yet reached clinical application. When trying to link viral foreignization to clinical applications, there is a problem that the virus that can be used is not toxic to living organisms and must be a heterogeneous virus to some extent for humans. There is a problem that it is difficult to make cancer foreign.

As a method for activating the immune system, there is a method other than the above-described method using a cancer cell as a foreign substance, by removing or suppressing the activity of an immunosuppressive factor originally present in a living body. Regarding this, it has been reported in animal experiments that administration of a small amount of an anticancer drug at an appropriate timing can result in activation of the immune system of a living body (Hosokawa, "Oncologia", 6:65, 1983). that is,
Suppressor T (T _s ), suppressor macrophage (Mφ _s ) where the administered anticancer drug is an immunosuppressive cell factor
And the immune serum factor immunoregulatory
α-globulin (IRA), immunosuppressive acidic protein (IA
P), ferrin, α-2 macroglobulin, etc. are suppressed (Hosokawa, Id.).

However, in a clinical setting, when administering an anticancer drug for the purpose of stimulating immunostimulation, it is actually extremely difficult to accurately judge the administration conditions such as the amount and timing of administration. For example, even if the same anti-cancer agent is administered under the same conditions, depending on the immune status of the host, it sometimes works in completely opposite directions, such as activating immunity at one time and suppressing immunity at another time.

[0014] Because of the many complicated and subtle problems described above, in a conventional method for enhancing immunity with an anticancer drug, various immune-related factors and immune activities interacting as a network, such as NK activity, T _s , Mφ
_A number of parameters such as _s , IRA, and IAP have to be measured at the same time, which requires a lot of time and labor. In addition, it is actually difficult to clearly determine the degree of immunostimulation, for example, the obtained results may be inconsistent between suppression-related factors and enhancement-related factors.

[0015] As described above, the conventional immunostimulation with an anticancer agent may actually cause immunosuppression depending on various conditions and the physiological condition of the host. From this aspect, the defense mechanism of the living body works. Therefore, the emergence of a measurement parameter capable of accurately and promptly determining the immunostimulation state sufficient to cause tumor shrinkage has been awaited.

[0016]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and based on a single parameter,
It is an object of the present invention to provide a method for positively promoting the conversion of cancer into foreign substances by a safe, simple and reliable method, and a method for determining the degree of cancer conversion into foreign substances. In addition, the use of these methods promotes the foreignization of cancer, thereby stimulating the immune system of the living body, and furthermore, performs a non-specific immunological treatment on the foreign cancerized cancer cells, thereby treating the cancer. It is an object of the present invention to provide a method for reinforcing cancer effects and more effectively reducing cancer.

[0017]

Means for Solving the Problems The present inventors have conducted intensive studies and found that HSP (= heat shock pr
otein; also referred to as a stress protein or a heat shock protein) is used as a single parameter for accurately grasping the degree of cancer cell xenogeneization sufficient to cause the shrinkage of the tumor by the action of the defense mechanism of the living body (host). I found that I got it .

That is, the present inventors have studied the immunity of the host.
Release from the state where both vesicular and humoral are suppressed,
In addition, aiming at the host defense mechanism as a further target
Targeting markers on cancer cells to facilitate
As a result, the expression of HSP can make cancer foreign
In order to complete the present invention,
Reached.

That is , according to the present invention, cancer cells are regressed.
Do not apply stress of a degree that does not
To be added to animals (excluding humans) by irradiation
Heat shock to cancer cells of the animal (excluding humans).
Animal, characterized in that it expresses
(Excluding humans) .

Further, according to the present invention, the intensity of the stress to the extent that cancer cells do not regress, in addition to the animals by anticancer drugs administered or radiation (excluding humans), then its
Heat-shock samples collected from other animals (excluding humans).
Click protein immunologically reactive with the immunoreactive substance is added, the resulting heat shock protein - from said immunoreactive substance conjugate by detecting the heat shock protein amount in the sample an animal (except for human ) For determining the degree of cancer cell xenogeneization in animals (however, humans
Exclude) is provided.

Furthermore, according to the present invention, the animal by cancer foreign substances degree determination method according to the above (excluding human) and determining the extent of foreign matter of cancer cells, the animal (however
And subjecting the animal ( excluding humans) to non-specific immunological treatment.

According to the present invention, it is possible to determine the extent of cancer cell xenogeneization sufficient to cause tumor shrinkage much more easily and quickly than in the conventional method. In addition, since a single parameter is used, interpretation of the obtained result is not arbitrary, and a reliable and simple judgment can be made.

Hereinafter, the present invention will be described in detail.

HSP is a protein expressed in cells by applying some stress to the cells, for example, heat, drugs, radiation and the like. Although there are various types, 90 families, 70 families, 60
Family and small molecule family.

HSPs are not normally expressed on cells,
Once expressed in cells, the body's defense mechanism recognizes the non-exposed material as a foreign object and starts attacking. In the case of cancer, cancer cells have this H
The expression of SP allows the cancer cells to be attacked by the host's own defense system. By the way, when this is expressed in normal cells, it causes an autoimmune disease.

HSP Targeted by Host Defense Mechanism
HSP73, because of its ease of appearance across species
That is, HSP having a molecular weight of 73 kDa is most desirable.

In the present invention, HSP is expressed in cells by applying a stress of such a degree that the cancer cells do not regress.

The means for applying stress to the cells is not particularly limited, but includes various known means including treatment means used in ordinary cancer treatment such as administration of anticancer drugs, irradiation, and heat curing. Can be used. For example, in the administration of an anticancer drug, any anticancer drug used may be used as long as it is commonly used in clinical practice or the like. For example, mitomycin C (MMC), endoxane (E
X), adriamycin (ADM), and fluorouracil (5-FU).

The term "stress having such a strength that the cancer cells do not regress" means that the cancer cells themselves do not regress when the stress is applied, and the HSP, preferably HSP, is added to the cancer cells.
It means the stress of the strength (weakness) of the minimum necessary to express 73.

By expressing HSP73 in cancer cells in this way, it is possible to cause cancer cells to become foreign,
That is, the antigenicity of the cancer cells is increased, and thereby immunostimulation of the living body can be achieved. The type of cancer is not particularly limited, and various types of malignant tumors can be applied.

Furthermore, in the present invention, an HSP produced by adding an immunoreactive substance immunologically reactive with HSP to cancer cells,
-A method for determining the degree of cancer xenogeneization, comprising determining the degree of xenogeneization of cancer cells by detecting the amount of HSP in the cells from the immunoreactive substance conjugate. In addition to the cancer cells, those tissues or extracts thereof, and samples collected from patients such as body fluids such as serum, urine, and cerebrospinal fluid can be used.

Examples of the immunological substance immunologically reactive with HSP, preferably HSP73, include, for example, anti-HSP7
3 antiserum, anti-HSP73 polyclonal antibody, anti-HSP
73 monoclonal antibody and the like. These can be used alone or in combination. In addition to these immunoreactive substances, it is also possible to detect the level of HSP73 in the sample by measuring HSP73 mRNA using, for example, a radioactive or non-radioactive labeled sense or antisense DNA probe. is there.

The method of adding an immunoreactive substance to cancer cells (a test sample derived from a patient), detecting HSP73 in the HSP73-immunoreactive substance conjugate produced, and measuring the amount of the HSP73 in the sample is described in Immunoreaction. An example in which the substance is an antibody will be described below.

Antibodies are proteins that have the ability to bind to and recognize other molecules known as antigens.
Monoclonal antibodies are not substantially different from other antibodies, but they are very homogeneous in their nature and recognize only one antigen or antigenic determinant. The antibody is prepared, for example, as follows.

First, an animal is immunized with HSP73,
The antibody producing cells are collected. As animals to be immunized, for example, experimental animals such as rats, mice, and hamsters can be advantageously used, and rats are particularly preferable. For example, in the case of rats, immunization can be performed by any route, such as subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal, but it is preferable to inject subcutaneously, intraperitoneally, or intravenously. Antibody producing cells
Splenocytes of the immunized animal are advantageously used. An established human spleen cell line may be used.

Next, the spleen cells and the myeloma cells of the obtained immunized animal are fused to obtain a hybridoma. In the present invention, cell fusion methods, conditions and the like can be according to a conventional method,
For example, Koehler and Milstein
n) et al. ("Nature", 256 , 495, 1975). A monoclonal antibody can be produced by screening the hybridoma by a conventional method and further cloning.

The antibody thus obtained is added to a test sample derived from a cancer patient to generate an HSP73-antibody conjugate. By recognizing HSP bound to the antibody by immunochemical measurement and measuring the amount thereof,
The HSP level in the sample can be known.

As an immunochemical assay, in principle,
All conventional immunoassays such as EIA, EL
An ISA method, an RIA method, or the like can be used. These immunochemical assays can generally be broadly classified into the following methods. (1) Competition method: A test solution containing an unknown amount of an antigen and a fixed amount of an antigen labeled with a labeling agent are subjected to a competitive reaction with a certain amount of a corresponding antibody to bind to the labeling agent bound to the antibody or to the antibody. The activity of the labeling agent not performed is measured. (2) Sandwich method: A test solution containing an unknown amount of antigen is reacted with an excess amount of antibody held on a carrier (first method).
Reaction), and then add a certain amount of an excess amount of the antibody labeled with a labeling agent to cause a reaction (second reaction). The activity of the labeling agent held on the carrier or the labeling agent not held on the carrier is measured. The first reaction and the second reaction may be performed at the same time or at different times. When the labeling agent is a radioisotope, the measurement is performed using a well counter or a liquid scintillation counter. When the labeling agent is an enzyme, the substrate is added and allowed to stand, and the enzyme activity is measured by a colorimetric method or a fluorescent method. Even if the labeling agent is a fluorescent substance or a luminescent substance, the measurement is performed according to a known method.

In addition to the above-mentioned methods, recently, Western blotting has been performed, in which an electrophoresed protein is transferred to a filter such as nitrocellulose and the target protein is detected using an antibody. Of course, it can be used for the detection of HSP73 in the present invention.

The antibody used in these assays can be labeled by a known antibody labeling method, and can be labeled with an appropriate marker such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like. . As the radioisotope, for example, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, ³⁵ S and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, glycosidase (eg, β-galactosidase, β-glucosidase, β-glucuronidase, β-fructosidase, α-galactosidase, α-galactosidase,
α-glucosidase, α-mannosidase), amylase (eg, α-amylase, β-amylase, isoamylase, glucoamylase, takaamylase), carbohydrase such as cellulase, lysozyme; amidase such as urease, asparaginase; cholinesterase (eg, acetyl Esterases such as cholinesterase), phosphatases (eg, alkaline phosphatase), sulfatase, and lipase; deoxyribonucleases;
Nucleases such as ribonuclease; iron and porphyrin enzymes such as catalase, peroxidase, and cytochrome oxidase; copper enzymes such as tyrosinase and ascorbate oxidase; alcohol dehydrogenase, malate dehydrogenase, lactate dehydrogenase, and isocitrate dehydrogenase Dehydrogenase is used. Examples of the fluorescent substance include fluorescamine and fluorescein isothiocyanate, and examples of the luminescent substance include luminol, a luminol derivative, luciferin, and lucigenin.

As a method for binding the antibody and the labeling agent,
Chloramine T method ("Nature", 194: 495,1962)
), Periodate method ("Journal of Histochemitry and C
ytochemistry ", 22: 1084,1974), maleimide method (" Jour
For example, the EIA method is performed as follows: First, a test sample is added to an antibody held on a carrier, and a binding reaction is performed. Then, an antibody conjugated with a labeling agent (for example, peroxidase) is added thereto, and the reaction is carried out.A substrate of peroxidase is added to the obtained reaction product, and the absorbance or the fluorescence intensity of the resulting substance is measured. The enzymatic activity of the reaction product is measured. The above series of operations is performed in advance on a known amount of a standard solution of the antibody reactant, and the relationship between the reactant and the absorbance or fluorescence intensity is created as a standard curve. Then, the absorbance or the fluorescence intensity obtained for the unknown amount of the analyte (test sample) is fitted to a standard curve, and the amount of the substance reacting with the antibody in the test sample is measured.

The RIA method is performed, for example, as follows. First, a test sample is added to the antibody held on the carrier to cause a binding reaction, and an antibody labeled with a labeling agent (for example, ¹²⁵ I) is added thereto and reacted. The γ-radioactivity of the obtained reaction product is measured. Performing a series of the above operations in advance on a known amount of a standard solution of the antibody reactant,
The relationship between the reactants and γ-radioactivity is prepared as a standard curve. Then, the γ-radioactivity obtained for the unknown amount of the analyte (test sample) is fitted to a standard curve, and the amount of the substance reacting with the antibody in the test sample is measured.

In addition to the method using an antibody, for example, HS
Of course, labeled antisense DNA oligonucleotides having a sequence complementary to P73 mRNA can be used. The antisense molecule has a sequence that is complementary or substantially complementary to a portion of the RNA transcribed from the selected gene, and
Form a double strand with A. Any oligonucleotide having sufficient complementarity to form a stable complex with the target RNA is deemed suitable. The antisense oligonucleotide may be complementary in substantially any region within the target RNA. The antisense molecule can be used as a DNA probe for detecting an increase or a decrease in mRNA expression specific to the HSP73 gene. That is, by specifically adhering to the target HSP73 mRNA to form a molecular hybrid,
The degree of HSP73 expression in the cells can be detected.

The oligonucleotide used can represent an unmodified oligonucleotide or an oligonucleotide analogue. Suitable analogs include, for example, ethyl- or methylphosphonate analogs, phosphorothioate-modified oligodeoxynucleotides ("Nucleic Acids Res.", 14: 9081, 1986; "J. Am. Chem.
Soc. ", 106: 6077, 1984. Further, recent advances in the production of oligonucleotide analogs include, for example, 2'-O-methyl ribonucleotides (" Nucleic Aci ").
ds Res. ", 15: 6131, 1987) and a chimeric oligonucleotide which is a complex RNA-DNA analog (" FEBS Lett. ", 2).
15: 327, 1987) can be used for the purposes described herein.

The selected oligonucleotide may be of any type, including charged and uncharged. Oligonucleotides of various lengths can be used, but those having about 8 to 40 bases are preferred. In order to perform such an experiment in vitro or in vivo, the oligonucleotide is labeled with a known labeling agent such as a radioisotope or a fluorescent substance by a conventional method. Examples of radioisotopes include ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴
There is a C, ³⁵ S and the like. Among them, the random primer method as a radioisotope ("Anal. Biochem.", 132: 6, 1983)
It is preferred to label with ³² P using Derivatively formed fluorescent dyes are also easier and less dangerous to handle. As the fluorescent dye, any dye that binds to an oligonucleotide can be used, but fluorescein, rhodamine, Texas Red, 4-fluoro-7-nitrobenzofurazan (NB
D), coumarin, fluorescamine, succinylfluorescein, dansyl and the like are preferably used.

Further, Northern blot analysis using cDNA can be performed as follows. That is, RNA prepared from a target cell or tissue is subjected to agarose gel electrophoresis, transferred to nitrocellulose or nylon membrane, and then reacted with a labeled HSP73 cDNA probe to measure HSP73 mRNA. The HSP73 cDNA probe used is HSP
It is desirable that the DNA be complementary to 73 mRNA and have a length of 17 bases or more.

In the present invention, the presence or absence of HSP73 in cancer cells (test sample) is detected as described above,
When the presence of SP73 is detected, HSP is added to the cancer cells.
Since 73 has been expressed, it can be determined that the substance has been converted into a foreign substance. That is, it is possible to easily and reliably determine the degree of cancer conversion into foreign matter using HSP73 as a single parameter. Then, when the presence of HSP73 is not detected, as described above, the HSP73 is expressed in the cancer cells by applying a stress of such a degree that the cancer cells do not regress, and is used as a standard for achieving cancer foreign matter. be able to.

When it is determined that HSP73 is expressed in the cancer cells by using the above-mentioned method, the cancer cells that have been made foreign can be effectively treated by non-specific immunological treatment. Can be reduced.

Here, the non-specific immunological treatment includes, for example, administration of non-specific immunotherapeutic agents such as zymosan, BCG, PSK and the like.

That is, the cancer is made foreign and the antigenicity of the cancer cells is increased, thereby activating the immune system. Then, by performing non-specific immunological treatment on cancer cells in such a state, the effect of this treatment will be reinforced and enhanced by the activated immune system, and cancer regression will be more effectively achieved. You can proceed.

One example of a specific method for expressing HSP73 in cancer cells by stress is to administer a small amount of an anticancer agent to the extent that the cancer does not regress itself. However, such a small amount of the anticancer agent may be administered. There is no danger of having significant side effects on the patient. Then, once HSP73 is expressed in this way to make cancer foreign,
Furthermore, the expression of the antitumor effect can be increased by stimulating and activating the level of the host defense mechanism by applying non-specific immunological treatment. That is, a greater effect of cancer regression can be obtained despite mild treatment.

According to the present invention, a strong antitumor resistance is imparted to a living body by converting a cancer into a foreign substance. Enhancing the antigenicity of cancer cells aims to place cancer cells, which have only weak and unstable antigens, into the field of an immune response and to enhance their immunological aggression against cancer. Non-specific immunotherapy of xenografted cancer cells against residual cancer and metastatic cancer after cancer treatment completely rejects the cancer-bearing organism. This is the ultimate goal of cancer foreignization.

[0053]

EXAMPLES The present invention will be described below in more detail with reference to Examples, which do not limit the scope of the present invention. Example 1 Preparation of Anti-HSP73 Clonal Antibody 0.2 ml of PBS (−) (Cosmo Bio, Cat. # 320-01) containing 160 μg of HSP73 was mixed with an equal amount of Freund's complete adjuvant (Yatron, Cat. # RM606-1). 0.2 ml of the resulting mixture was subcutaneously administered to Lurat (5-week-old, female) (CLEA Japan) to immunize.
Ninety days after the second and third immunizations were repeated in the same manner, spleens were removed from the immunized rats. The adipose tissue attached to the spleen was removed, and Hanks balanced salt solution (GIBCO, Cat. # 3
The cells were loosened with forceps in 10-4170PJ) and filtered through a stainless steel mesh. The cells thus collected are added to a Tris-buffered ammonium chloride solution 10
Erythrocytes were destroyed and removed by adding ml. Tris-buffered ammonium chloride solution is prepared by dissolving 20.594 g of trishydroxymethylaminomethane in about 800 ml of purified water.
After adjusting the pH to 7.65 with N-hydrochloric acid, the solution was adjusted to a total volume of 1 liter with purified water to obtain solution A. One volume of this solution A was 0.83%
The solution was added to 9 volumes of ammonium chloride solution, sterilized by a Millipore filter, and prepared.

The spleen cells of the immunized rat 1.38 × 10
^{Eight of} them were RPMI1640 medium (GIBCO, Cat. # 320-1875PJ).
) Disperse in 1 ml and likewise in RPMI 1640 medium
Rat myeloma cells Y3-Ag 1.2.
3. (Dainippon Pharmaceutical, Cat. # 09-1631) 2.7 × 10 ⁷ cells (at a cell ratio of 5: 1), and a 50% polyethylene glycol 1500 solution (Boehringer Mannheim Yamanouchi Cat. # 783641) ) Add 1 ml and add RPMI164
0 ml of medium 0 was added and left at 37 ° C. for 5 minutes to perform cell fusion.

After the fusion, the cells were washed and incubated at 37 ° C. in HAT medium [MEM Dulbecco's solution (Dainippon Pharmaceutical, Cat. # 12-333-5).
4) 1 g of kanamycin sulfate (Meiji Seika) in 200 ml
Is dissolved in 40 ml of Hanks (Dainippon Pharmaceutical, Cat. # 12-132-54), and 0.8 ml of glutamine (Nissui Pharmaceutical, Ca
# 05908) 2 ml of 0.3 g dissolved in 10 ml of Hanks, 100 mM pyruvate (Dainippon Pharmaceutical, Cat. # 1
6-820-49) 2 ml, 10 mM β-mercaptoethanol (Seikagaku Corporation, Cat. # 137-06862) Hank's solution 1 m
1, HAT 50 times solution (Dainippon Pharmaceutical, Cat. # 16-808-49) 4
ml, fetal calf serum (Sera-Lab., Cat. # S-0001a) 30 ml
20 ml of BM-Condimed (Boehringer Mannheim Yamanouchi, Cat. # 1088 947)
What was added] was 98 ml, and the cells were well suspended. One plate was added to four 24-well plates for cell culture (Falcon, Cat. # 3047).
The mixture was dispensed at a rate of ml / well, and culture was started at 37 ° C. in a CO ₂ incubator. One week later, each well contained an HT medium [having the same composition as the HAT medium described above, and HT50 (Dainippon Pharmaceutical, Cat. # 16-809-49) was used for 1/50 instead of HAT50.
Was added, and the cells were cultured for one week. The proliferation of the hybridoma was observed under a microscope, and the antibody titer in the culture supernatant of each well was measured by ELISA. Of the cells in the wells with high antibody titers, BALB / 3T3 cells / lysa were analyzed by Western blotting.
The cells in the wells with high reactivity with HSP73 in te were cloned by the limiting dilution method. That is, the cell dispersion was transferred to a cloning medium [HAT
Medium, except for HAT50), and spread the cells on a 96-well plate (Falcon, Cat. # 3072) so that the number of cells would be 0.5 cells / well on average, and cultured in a cloning medium. Regarding the culture supernatant of the well in which cell proliferation was observed, clones having high reactivity among clones confirmed to be reactive with HSP73 were cloned again by the limiting dilution method, and the antibody secretion ability was high. An antibody-producing hybridoma was established by selecting clones having excellent growth properties and being stable. This hybridoma is HSP
Production of an antibody that specifically reacts with 73 was confirmed by the following ELISA method and Western blot method.

This hybridoma (cell number 3.65 × 1)
0 ^6), 175T cell culture flask (culture surface area 1
Culture medium [MEM Dulbecco's solution (Dainippon Pharmaceutical, Cat. # 12-333-54) prepared at 75 cm ² ) for hybridoma culture.
0.8 ml of a solution of 1 g of kanamycin sulfate (Meiji Seika) in 40 ml of Hanks (Dainippon Pharmaceutical, Cat. # 12-132-54) and 200 ml of glutamine (Nissui Pharmaceutical, Cat. # 0)
5908) Dissolve 0.3 g in 10 ml Hanks and add 2
ml, 100 mM pyruvate (Dainippon Pharmaceutical, Cat. # 16-82
0-49) 2 ml, 1 mM Hank's solution of β-mercaptoethanol (Sigma, Cat. # M-6250), 1 ml of fetal calf serum (Sera-Lab. Cat. # S-0001a) 30 ml) The resulting suspension was dispensed into tissue culture flasks at 25 ml each and cultured at 37 ° C.
On the fourth day of the culture, the culture supernatant was collected, separated from the antibody fraction by ammonium sulfate salting out, and then purified by an antibody affinity column by a conventional method to obtain an anti-HSP73 monoclonal antibody.
This anti-HSP73 monoclonal antibody was obtained from Rat
Mono AB ID / SP Kit (Zymed, Cat. # 9
3-9550) by immunoglobulin class measurement
It was confirmed to be an IgG class immunoglobulin. Evaluation of anti-HSP73 monoclonal antibody characteristics (1) Enzyme antibody method (ELISA method) HSP73 was treated with 0.05M sodium carbonate at pH 9.6.
It was dissolved in a sodium bicarbonate buffer (CB buffer) to a concentration of 0.625 μg / ml, and 100 μl was added dropwise to each well of an immunoplate (Nunc, Cat. # 439454). The outermost well was filled with CB buffer, left overnight at 4 ° C. in a humid atmosphere, and then the antigen solution was discarded.
BS-Tween (Tween-20 0.05% (v
/ V), and each well was washed with a solution dissolved in PBS (-). 300 μl of PBS-T per well
Wean was added and left at room temperature for 3 minutes.
After washing three times with Ween, 150 μl of the sample (the anti-HSP73 antibody IgG: primary antibody) was added to each well, left at room temperature for 2 hours, and then the plate was washed six times with PBS-Tween. Thereafter, 200 μl of a secondary antibody (peroxidase-labeled anti-rat IgG) was added to each well, allowed to stand at room temperature for 2 hours, and washed 9 times with PBS-Tween. OPD
(O-Phenylenediamine) (Sigma, Cat. # P8287) 10mg
One of them was added with 5 μl of aqueous hydrogen peroxide at 0.1 M pH5.
0 μl of a substrate solution dissolved in 12.5 ml of citrate phosphate buffer was added dropwise to each well, and protected from light at room temperature.
After standing for 0 minutes, 50 μl of 12.5% (w / w) sulfuric acid was added to each well to develop a color, and the 492 nm O.D. D. To a microplate reader (Tosoh MPR-
(A4i type). (2) Western blot method Laemmli buffer system (Laemmli, NK, "Nature", 283 : pp. 249-256, 1970) by SDS-PAGE method.
The lysate electrophoresis of BALB / 3T3 cells was carried out at. The concentrated gel used had the following composition. 6.1 ml of distilled water, 0.5 M Tris (Bio-Rad, Cat. # 161-0
716) -HCl (pH 6.8) 2.5 ml, 10% SDS
(Bio-Rad, Cat. # 161-0301) 100 μl, 30% acrylamide (Bio-Rad, Cat. # 161-0101) / bis (Bio-Rad, Cat. # 161-0301)
# 161-0201) Mix 1.3ml and degas for at least 15 minutes,
10% ammonium persulfate (Bio-Rad, Cat. # 161-0700) 5
0 μl, TEMED (Bio-Rad, Cat. # 161-0800) 10 μl
Was added. Separation gels were prepared as follows.
4.045 ml of distilled water, 1.5 M Tris-HCl 2.5
Slowly mix 100 ml of 10% SDS and 3.3 ml of 30% acrylamide / bis and degas with an aspirator for at least 15 minutes.
l, 5 μl of TEMED were added. As an electrophoresis buffer, 9.0 g of Tris and glycine (Bio-Rad, Cat. # 161-071) were used.
7) Add distilled water to 43.2 g and 3.0 g of SDS, and add 600 m
and diluted 5-fold with distilled water. The sample buffer was distilled water 2 ml, 2M Tris-HC
1 (pH 6.8) 500 μl, SDS 0.32 g, β-
800 μl of mercaptoethanol, 0.05% (w /
v) Bromophenol blue (Bio-Rad, Cat. # 161-0404)
What mixed 400 microliters was used. After that, Trans-Bl
0.45 μm nitrocellulose membrane (Schleicher & Schuell, Cat. # 401196) at room temperature and 100 V using an ot Electrophoretic Transfer Cell (Bio-Rad).
The gel was adhered to the sample, and blotting was performed at 4 ° C. for 3 hours. Tris Gly R as blotting buffer
unning and Blotting Buffer (Enprotech, MA., USA, C
at.No.SA100034) 20% MeOH was used.

After that, 5% skim milk (snow mark) -PB
It was immersed in the S (-) solution at room temperature for 30 minutes to perform blocking. After blocking, a primary antibody reaction was performed using a culture supernatant of the hybridoma as a primary antibody using a screener blotter (SAMPLATEC, Inc.). Thereafter, the solution was changed twice with PBS (-) for 5 minutes,
Washing with slow rocking shaker and PBS-
The solution was replaced with a 0.1% Tween 20 (Bio-Rad, Cat. # 170-6531) solution for 15 minutes, and four washes were performed. Finally, two washes were performed with PBS (-) for 5 minutes.

After the washing was completed, a peroxidase-labeled goat anti-rat IgG antibody (Southern Biotechnology, Cat. # 3030)
-05) with 5% BSA (Seikagaku Corporation, Cat. # 250020) -PB
After diluting 500-fold with the S (-) solution, a secondary antibody reaction was performed for 2 hours. After the reaction is completed, the membrane is
(-) The solution was changed twice with PBS-0.
Five washes were performed with a 1% Tween 20 solution for 15 minutes while changing the solution, using a slow rocking shaker. Finally, washing was performed twice with a PBS (-) solution for 5 minutes. After removing the excess PBS (-) solution, ECL Western bl
Sprinkle otting detectionreagent (Amersham, Cat. # RPN2106) on the membrane, incubate for 1 minute, remove excess detection reagent, wrap the membrane in wrap, and place the reaction surface on X-ray film (Kodak X-OMAT, AR Cat). . #
165 1454), exposed, developed, and
The reactivity of the anti-HSP73 monoclonal antibody was examined by measuring a band near the molecular weight of 73 kDa corresponding to the above. Example 2 Detection of HSP73 on Sarcoma 180
Outbreak and cancer treatment effects 4-5 week old ICR-JCL mice (females) were divided into four groups, the first group was used as a control group (10 animals),
Groups (12 each) were treated with mitomycin C (MM
C) Administration group, MMC + PSK (intraperitoneal) administration group, MMC
+ PSK (oral) administration group. Then, for each of these four groups, 1 × 10 ⁶ Sarcoma 180 cells
Individuals were implanted intraperitoneally.

[0059] Mice of the second to fourth groups, transplant day than 4 days, 1 / 2LD ₅₀ amount MMC 1.95 mg / kg
Was administered daily by intravenous injection. After one week of drug holiday, dosing was continued for another four days. In addition to the third and fourth groups, daily PSK was 10 mg / k from the day after transplantation.
g was administered intraperitoneally (Group 3) or 1 g / kg orally (Group 4).

On the 10th day after transplantation, MMC was administered on the second to second days.
Two animals from each of the four groups were treated with H by the following method.
The presence or absence of SP73 was examined.

Specifically, tumor cells were aseptically removed from each mouse, washed in a 1.5 ml tube with PBS, homogenized with a Teflon homogenizer, and then lysis buffer [NP-40 1.0%, NaCl
0.15 M, Tris-HCl (pH 8.0) 50 m
M, EDTA 5 mM, N-ethylmaleimide 2 m
M, PMSF 2 mM, leupeptin 2 μg / ml,
2 μg / ml of pepstatin] and add 2 ml on ice.
Incubated for 0 minutes. Thereafter, the cells were detached with a scraper, collected in a 1.5 ml tube, and heated at 4 ° C. /
Centrifugation was performed at 12000 rpm for 20 minutes. PB
10 μl of the supernatant after centrifugation was added to 790 μl of S (−), and Dye Reagent Concentrate (Bio-Rad, C
at 500 ° -0006), incubated for 5 minutes at room temperature, measured the absorbance at 595 nm, quantified the protein, and used as a sample for electrophoresis.

Using the above sample, the SD described in Example 1 was used.
When the presence or absence of HSP73 was examined by S-PAGE, the appearance of HSP73 was observed in Sarcoma 180 cells of all individuals.

FIG. 1 shows the survival rate of each group. As is clear from FIG. 1, all of the control group died on the 16th day after transplantation, and all of the MMC-only administration groups died on the 20th day after transplantation. However, in the MMC + PSK (intraperitoneal) administration group, all survived by 16 days after transplantation, and all died only 23 days after transplantation. Also, MM
In the C + PSK (oral) administration group, about 20% survived 30 days or more after transplantation. In other words, it was found that the combined use of the administration of MMC in such an amount that the cancer does not regress alone and the administration of PSK, which is a nonspecific immunotherapeutic agent, can provide a remarkable life-extending effect of the cancer-transplanted mouse. Example 3 Detection of HSP73 in L-1210 cells
And divided into four groups of BDF ₁ mice (male) of cancer treatment effect 6-7 weeks old, the first group and the control group (10 mice), adriamycin second to fourth groups (each each 12 mice), respectively (ADM ) Administration group, ADM + PSK (intraperitoneal) administration group, ADM + PS
K (oral) administration group. Then, for each of these four groups, 1 × 10 ⁶ L-1210 cells were transplanted intraperitoneally.

[0064] In the mouse of the second to fourth group, on the sixth day after the transplantation and transplant the next day, a 1 / 2LD ₅₀ the amount of the ADM 11.15m
g / kg was administered by intravenous injection. In addition to the third and fourth groups, 10 mg PSK every day from the day after transplantation
/ Kg intraperitoneal administration (Group 3) or 1 g / kg
Was orally administered (group 4).

On the 10th day after transplantation, ADM was administered on the second day.
When the presence or absence of HSP73 was examined based on the method described in Example 2 for two animals from each of the four groups, the appearance of HSP73 was observed in L-1210 cells of all individuals.

FIG. 2 shows the survival rate of each group. As is clear from FIG. 2, all of the control group died on the 8th day after the transplantation, and the survival rate on the 25th day after the transplantation was about 50% in the ADM only administration group. However, ADM + PSK
In both the (intraperitoneal) administration group and the ADM + PSK (oral) administration group, 70 to 80% of the individuals survived 25 days after transplantation. That is, in the group in which HSP73 appeared by ADM administration, it was found that a remarkable life-prolonging effect was obtained by using the nonspecific immunotherapeutic agent PSK together as compared with the case of ADM administration alone. Example 4 of HSP73 on Sarcoma 180
Detection and cancer treatment effect ICR mice (females) of 4-5 weeks old were divided into three groups,
The group was a control group (10 animals), and the second and third groups (1 each)
2 animals each), irradiation group, irradiation + PS
K (oral) administration group. Then, for each of these three groups, 1 × 10 ⁶ Sarcoma 180 cells were implanted subcutaneously in the thigh.

Two weeks after transplantation, the second and third groups of mice were locally irradiated with 15 GY of radiation (single irradiation). For the third group, 1 g of PSK every day from the day after transplantation
/ Kg orally.

The second and third irradiations were performed on the seventh day of irradiation.
When two animals from each group were examined for the presence or absence of HSP73 based on the method described in Example 2,
HSP was added to Sarcoma 180 cells of each individual
73 appearances were observed.

FIG. 3 shows the results of treatment in each group. FIG.
As is clear from the above, in the group in which HSP73 appeared by irradiation, the tumor was reduced by further PSK administration.

[0070]

As described above in detail, according to the present invention, the administration of an anticancer drug is carried out by applying a stress having such a strength that the cancer cells do not regress.
Or HS by adding to cancer cells by irradiation
By expressing P, it can make cancer foreign and increase cancer antigenicity, thereby activating the immune system. Also, HS
By using an immunoreactive substance immunologically reactive with P, the amount of HSP expression can be measured safely and efficiently.

In this way, the degree of foreign body formation is determined based on the expression of HSP in cancer cells, and the level of the biological defense mechanism is reduced by subjecting such foreign body cancer cells to non-specific immunological treatment. By stimulating and activating, the expression of the antitumor effect can be increased. That is,
Greater effects of cancer regression can be obtained despite mild treatment. Thus, the clinical significance of the present invention is extremely high.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of treating cancer with Sarcoma 180 cells in mice.

FIG. 2 is a graph showing the effect of treating cancer with L-1210 cells in mice.

FIG. 3 is a graph showing the effect of treating cancer with Sarcoma 180 cells in mice.

 ──────────────────────────────────────────────────続 き Continued on the front page (56) References MEDICAL IMMUNOLOG Y, VOL. 24, NO. 4, (1992), p. 309-316 INT. J. HYPERTHERMI A, VOL. 2, NO. 3, (1986), p. 267-275

Claims (3)

(57) [Claims] Claims: 1. An animal (provided that an anticancer drug is administered or radiation is applied to the animal (however,
By adding to animals ( excluding humans) ,
A method for promoting carcinogenesis in animals (excluding humans), which comprises expressing a heat shock protein in cancer cells (excluding humans) . 2. An animal (provided that an anticancer drug is administered or radiation is applied to the animal (however,
In addition to humans) , the animals (except humans)
Samples taken from excluding) the immune reactants with heat shock proteins immunologically reactive addition, the resulting heat shock protein - from said immunoreactive substance conjugate by detecting the heat shock protein mass in the sample Animals (however,
A method for determining the degree of cancer xenogeneization of animals (excluding humans), comprising determining the degree of xenogeneization of cancer cells (excluding humans) . 3. Animals by cancer foreign substances degree determination method according to claim 2 (excluding human) and determining the extent of foreign matter of cancer cells, the animals (except for human) Subjecting the animal to a non-specific immunological treatment .
(Excluding humans) .