JPH11139994A - Antitumor agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antitumor agent capable of extremely effectively controlling a cancer, by using a complex of a gene encoding interleukin (IL-18) and a Iiposome to insert a gene encoding IL-18 into the interior of a cancerous cell. SOLUTION: This antitumor agent contains a complex of (A) a gene encoding interleukin IL-18 or a gene containing a sequence in which an amino acid is deleted, substituted or added in the amino acid sequence of IL-18 and having IL-18 activity and (B) a liposome. An oligodeoxyribonucleotide (DNA) is preferable as the component A. A bond between the nucleotides is preferably a phosphorothioate type bond. A cationic liposome is preferable as the component B. The shape of the complex is preferably an inclusion complex. The administration form is preferably an injection which can be applied to a local part of a tumor. The antitumor agent preferably contains 1-10 wt.% of a gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antitumor agent.

[0002]

2. Description of the Related Art Interleukin-18 (Interl)
eukin-18; abbreviation = IL-18) was identified in 1995 by Okamura et al. in the liver tissue of a mouse fulminant hepatitis model [Nature, 378 , 88-91 (199).
5)]. IL-18 acts on T cells and NK cells and
Since it has FN-γ production inducing activity, it was initially discovered that γ-
Interferon inducing factor (IFN-γ Induci
ng Factor; abbreviation = IGIF). Expression of IL-18-producing cells is observed not only in macrophages but also in skin keratinocytes and adrenal cortex cells [J. Biol. Chem. , 272 ,
26595-26603 (1997)]. It is also known that the synergistic effect with interleukin-12 (abbreviation = IL-12) further enhances the ability to induce production of γ-interferon (abbreviation = IFN-γ) [Eur.
J. Immunol. 26, 1647-1651 (19
96)]. Furthermore, it is similar in structure to interleukin-1β (abbreviation: IL-1β), and IL-1β converting enzyme (IL-1β converting enzyme;
It is also known that the active form is obtained by processing by the abbreviation = ICE (Nature, 386 , 619).
-623 (1997)].

[0003] In addition to the activity of inducing IFN-γ production, IL-18 has an effect of enhancing the expression and function of Fas ligand in lymphocytes [Clinical Immunity, 29 (6),
683-689 (1997)], or having the effect of inducing the production of granulocyte macrophage colony stimulating factor (abbreviation: GM-CSF) [J. Immuno
l. , 156, 4274-4279 (1996)]. Regarding the antitumor effect of IL-18, ascites-type Meth A sarcoma tumor-bearing mice were tested for IL-
It has been reported that systemic administration of 18 (protein) has the effect of reducing tumor size [Cancer
Immunol Immunother, 43, 36
1-367 (1997)]. However, there is no report on other antitumor activities, especially on solid tumors.

[0004]

SUMMARY OF THE INVENTION The present inventor has proposed IL-1
It has been found that cancer can be extremely effectively suppressed by inserting the gene encoding 8 into cancer cells. The present invention is based on these findings.

[0005]

SUMMARY OF THE INVENTION Accordingly, the present invention provides
(1) (a) a gene encoding interleukin-18, or (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of interleukin-18, and A gene encoding a protein having a leukin-18 activity (hereinafter sometimes referred to as an interleukin-18 variant or an IL-18 variant) and (2) a liposome complex. It relates to an antitumor agent.

[0006] As used herein, “anti-tumor activity” refers to
It refers to an action of preventing or suppressing the development, maturation, proliferation, or spread of tumor cells or tissues, or an action of regressing tumor cells or tissues. The tumor includes, for example, carcinoma or sarcoma. Whether or not a compound has the antitumor activity can be determined, for example, by the following method. When an experimental animal (eg, mouse) implanted with a solid tumor is used, a test compound is administered to the tumor-bearing animal for a certain period of time, and then a test compound-administered group and a control group (that is, non-administered test compound). Group) and tumors were excised, and their tumor weights were measured. The tumor weight was determined by whether a significant weight difference was observed between the tumor weight excised from the administration group and the tumor weight excised from the control group. , Have anti-tumor activity. In addition, instead of or in combination with the above-mentioned measurement of tumor weight, by comparing the qualitative change of cancer cells caused by the pathological findings of the resected tumor foci, and / or prolonging the life expectancy Comparing the rates can also determine whether they have anti-tumor activity. Alternatively, when an experimental animal implanted with ascites cancer is used, the test compound is administered to the tumor-bearing animal for a certain period of time, and then the test compound-administered group and the control group (that is, the test compound non-administered group) are used. Was collected from each sample, and the number of cancer cells contained in the ascites was measured. A significant difference was found between the number of cancer cells in the ascites fluid of the administration group and the number of cancer cells in the ascites of the control group. Can determine whether it has antitumor activity.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The complex used as an active ingredient in the antitumor agent according to the present invention contains the above gene and liposome. As the above-mentioned gene, a gene encoding IL-18 can be used. IL-18 can be obtained from various animals, particularly mammalian (eg, human) IL-18, eg, human IL.
-18 [amino acid sequence is described in J. Immunol. , 15
6 , 4274-4279 (1996)], or mouse IL-18 [amino acid sequence is described in Nature, 37
8 , 88-91 (1995)]. As the above-mentioned gene, a gene encoding an IL-18 variant can also be used. The IL-18 variant comprises an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of IL-18, and has an interleukin-18 activity (particularly an antitumor activity). Protein.

Each of the above genes can be used alone or in combination of two or more. Each of the above genes can be a gene encoding a mature form or a gene encoding a precursor. Also,
It may be a gene encoding a fusion protein of IL-18 or a variant of IL-18 and another appropriate protein (eg, IL-12, ICE, etc.).

The above-mentioned gene is formed from deoxynucleosides, ribonucleosides, and / or modified ribonucleosides thereof (for example, 2'-O-modified ribonucleosides, preferably 2'-O-methylribonucleosides). be able to. Therefore, the genes used in the present invention include ribonucleotides (RNA) consisting of ribonucleosides and / or modified ribonucleosides, deoxyribonucleotides (DNA) consisting only of deoxyribonucleosides, or ribonucleosides (and / or modified ribonucleosides) and deoxyribonucleosides. And an oligodeoxyribonucleotide (DNA) consisting solely of deoxyribonucleosides.

[0010] In the gene used in the present invention, the internucleotide bond between each nucleoside can be independently a phosphodiester bond or a modified phosphodiester bond. Examples of the modified phosphodiester bond include a methylphosphonate-type bond in which one oxygen atom of two non-crosslinking oxygen atoms of a phosphodiester bond is substituted with a methyl group, and two non-crosslinking oxygen atoms of a phosphodiester bond A phosphoramidate-type bond in which one oxygen atom is substituted with an amino group or a substituted amino group, and a phosphorothioate type in which one oxygen atom of two non-crosslinked oxygen atoms in a phosphodiester bond is substituted with a sulfur atom Or a phosphorodithioate-type bond in which two oxygen atoms of two non-bridging oxygen atoms of a phosphodiester bond are substituted with two sulfur atoms, and one or more of these are bonded. , One of the bonds between nucleosides
It can be introduced at a point or more. By including a modified phosphodiester bond at one or more sites, base sequence specificity, duplex stability, antinuclease resistance, cell membrane permeability, low cytotoxicity and moderate metabolism, and A simple preparation method and the like can be improved. Phosphorothioate-type bonds are particularly preferred because of their good stability in vivo. Further, it is particularly preferable that at least half (particularly all) of the bonds between nucleosides are modified phosphodiester bonds (particularly, phosphorothioate type bonds).

[0011] The gene used in the present invention can be prepared by a known method. For example, although not limited thereto, appropriate primers can be designed and prepared from a known nucleotide sequence, and a target gene can be obtained from an appropriate library by a PCR method or the like.

The liposome which can be used in the present invention is, for example, a liposome prepared from a lipid molecule such as a phospholipid, a glycolipid or cholesterol, and a single-layer liposome or a multi-layer liposome is effective.
Phospholipids from which liposomes can be prepared include:
Generally, for example, glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, or cardiopine) or sphingolipids (sphingomyelin, ceramide phosphorylethanolamine, or ceramide phosphorylglycerol) Can be mentioned. Examples of glycolipids from which liposomes can be prepared include, for example, glyceroglycolipid (digalactosyl diglyceride or seminolipid) or glycosphingolipid (galactosylceramide or lactosylceramide).

[0013] The liposome depends on the state of the electric charge of the polar part.
Examples include neutral liposomes, cationic liposomes, anionic liposomes, and pH-sensitive liposomes. Cationic liposomes are preferred because one component of the complex, the gene, is negatively charged, and the surface of the tumor cells is also negatively charged. For example,
Cationic liposomes are artificial mixtures of cationic head lipids and helper lipids, such as (1) (a) a monocationic head, such as D
OTMA {ie, N- [1- (2,3-dioleyloxy) propyl] -N, N, N-triethylammonium}, DMRIE {ie, N- [1- (2,3-
Dimyristyloxy) propyl] -N, N-dimethyl-
N- (2-hydroxyethyl) ammonium bromide {DOTAP}, ie, 1,2-dioleyloxy-3- (trimethylammonio) propane}, DDA
B {ie, dimethyldioctadecyl ammonium bromide}, PC-cholesterol {ie, 3β-
[N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol {or DOTIM}, ie, 1- [2-9 (Z) -octadecenoyloxy)
-Ethyl-2- (8 (Z) -heptadecenyl) -3-
(2-hydroxyethyl) -imidazolinium chloride｝, or (b) a polycationic head such as DO
GS {i.e., dioctadecylamidoglycylspermine} or DOSPA {i.e., 2,3-dioleyloxy-N- [2- (speminecarboxamido) ethyl] -N, N-dimethyl-1-propaneammonium trifluoroacetate And (2) a helper lipid such as DOPE {ie dioleoyl-phosphatidylethanolamine} or DOPC {ie.
Cationic liposomes consisting of dioleylphosphatidylchlorine II can be used.

In the antitumor agent according to the present invention, the above-mentioned gene and liposome are simultaneously complexed.
As long as they are contained in the form of x), their existence forms are not particularly limited. Examples of the complex include a mixture of the gene and liposome, and an embedded or encapsulated gene by the liposome, and an embedded body is preferable. The above-mentioned complex is, for example, a method of forming a complex using a gene and a liposome by using an electrostatic bond, that is, a method called a lipofection method, and the two are slowly mixed in a test tube and then left. Can be prepared by

The above-mentioned embedded body can be prepared, for example, by a method in which a gene is encapsulated in a liposome. That is, using a lipid such as phosphatidylserine, a multi-layer liposome is prepared by a vortex mixer or the like, and then subjected to ultrasonic treatment to prepare a single-layer liposome. The gene can be prepared by adding the gene to the obtained monolayer liposome, gently vortexing the mixture, and incubating at about 37 ° C. for about 10 minutes, or lyophilizing and rehydrating. The above encapsulated product can also be prepared by a known method.

The antitumor agent of the present invention contains a complex of the gene and the liposome as an active ingredient, and if necessary, contains a usual carrier which is pharmaceutically or veterinarily acceptable. Can be. The dosage form of the antitumor agent of the present invention is not particularly limited, for example, powders,
Oral preparations such as fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts and pills, or injections, external solutions, ointments, suppositories, and topical creams Or parenteral preparations such as eye drops, and particularly preferable are drugs administered locally to the tumor (eg, injections or sustained-release pellets).

The oral preparations include, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid ester, talc Excipients, such as magnesium stearate, polyethylene glycol, magnesium silicate, silicic anhydride, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, and preservatives Agents, coloring agents,
It can be produced according to a conventional method using a flavor, a flavoring agent, a stabilizer, a humectant, a preservative, an antioxidant and the like.

Examples of the parenteral administration method include injection (subcutaneous, intravenous, etc.) and rectal administration. Of these, injections are most preferably used. For example, in the preparation of an injection, in addition to the complex as an active ingredient,
For example, water-soluble solvents such as saline or Ringer's solution, water-insoluble solvents such as vegetable oils or fatty acid esters,
An isotonic agent such as glucose or sodium chloride, a solubilizer, a stabilizer, a preservative, a suspending agent, an emulsifier, or the like can be optionally used.

The antitumor agent of the present invention can be administered using a sustained-release preparation technique using a sustained-release polymer or the like. For example, the antitumor agent of the present invention can be incorporated into a pellet of ethylene vinyl acetate polymer and the pellet can be surgically implanted into the tissue to be treated.

The antitumor agent of the present invention is not limited to the active ingredient.
It can be contained in an amount of 0.01 to 10% by weight, preferably 0.1 to 10% by weight, more preferably 1 to 10% by weight.

The antitumor agent of the present invention can be administered to animals, preferably mammals (particularly humans). The dose when the antitumor agent of the present invention is used depends on the type, age, sex, or weight of the animal to be administered, the type of disease, the type of administration, or the like, and is not particularly limited. 1 μg / kg to 10000 per adult
μg / kg, preferably 10 μg / kg to 1000 μg
/ Kg, more preferably 50 μg / kg to 500 μg /
The dose of about kg is orally or preferably parenterally (particularly, topically) in about 1 to 4 times a day.

The antitumor agent of the present invention can be applied to any tumor in animals, preferably mammals (especially humans), but is particularly preferably cancer, preferably solid cancer, for example, head and neck cancer, It can be effectively applied to treatment of skin cancer, malignant melanoma, brain tumor and the like.

[0023]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 In this example, a mouse spleen cDNA library [Clontech Lad.
b. Inc. , Palo Alto, California, USA]
Was isolated from mouse IL-18 cDNA. Mouse IL
Based on the -18 (IGIF) cDNA sequence, primers were designed as follows. Forward primer: 5'-CACAAGCTTGGAACAATGGCTG
CCATGTCAGAA-3 ′ Reverse primer: 5′-GTGGGATCCCCCTAACTTTGATG
TAAGTTAGTGGAGAG-3 '. The PCR operation was performed at 94 ° C. for 1 minute, 55 ° C. for 45 seconds, and 72 ° C. for 2 minutes as one cycle.
35 cycles were performed using a thermal cycler (Perkin Elmer, USA). The PCR product was gel-extracted and the extract was cloned into a plasmid (pCR
2.1; Invitrogen Corp. , San Diego, CA, USA), and the competent JM109 cells (Takara Shuzo, Kyoto, Japan) were transformed with the recombinant plasmid. After ampicillin-resistant colonies were selected and cultured overnight in an LB medium, plasmid DNA was prepared by the alkaline PEG method, and the nucleotide sequence was confirmed by an automatic sequencer (Perkin Elmer, USA).
Eight mature cDNA was inserted.

[0024]

Example 2 In this example, mouse IL-18cDN
A was transfected into Lewis lung carcinoma cells in the form of an embedding with liposomes. Plasmid DN prepared in Example 1
A is cleaved with restriction oxygen HindIII and BstXI,
Vector pRC / CMV for animal cell expression (Invitr
oogen Corp. , San Diego, Calif., USA) and inserted into the E. coli HB101 strain (Takara Shuzo, Kyoto, Japan), followed by IL-18 expression plasmid DNA.
Was prepared. 10% Lewis lung cancer cells (LLH) (ATCC CRL1642) in dishes (diameter = 10 cm)
RPMI containing fetal calf serum (FCS) (GIBCO BRL, Gaithersburg, MD, USA)
After overnight culture in 1640 medium (GIBCOBRL, Gaithersburg, MD, USA), Opt
After washing 5 times with i-MEM (GIBCO BRL, Gaithersburg, MD, USA), 5 ml of Opti-MEM was finally placed.

To 5 μl (5 μg) of the above expression plasmid, 45 μl of sterile water and lipofectin (GIBCO BRL)
Co., Gaithersburg, MD, USA) 50μ
was added and mixed, and then allowed to stand at room temperature. After 15 minutes, the mixture was added to the dish containing Lewis lung cancer cells and 37
The cells were cultured in an incubator at 5 ° C. in the presence of 5% CO ₂ . After 5 hours, 5% RPMI1640 containing 10% FCS was added.
The cells were cultured with the addition of ml. The next day, neomycin (G41
8; GIBCO BRL, Gaithersburg, Md., USA) to a final concentration of 100 μg / ml, and 10 days later, G418 that formed a colony
A resistant clone strain was obtained. 10 colonies were trypsin / E
DTA solution (GIBCO BRL, Inc., Gaithersburg, MD, USA) were harvested by culturing the respective colonies derived G418 resistant strain (cells), after extraction of RNA by a conventional method, ³² P labeled IL-18cRN
Northern blotting was performed using A. Thus I
IL-1 expressing the L-18 gene at the highest level
8 Gene Transfer Lewis Lung Cancer Cell Line (LLH / IL-18)
Was selected.

[0026]

Example 3 In this example, mice were transfected with IL-18-introduced-Lewis lung cancer cell line (LLH / IL-18), and the antitumor activity was examined. IL selected in Example 2 above
-18 introduction-Lewis lung cancer cell line (LLH / IL-18)
Was further cultured in RPMI1640 medium containing 10% FCS, and then C57BL / 6 mice (8 weeks old, male)
Nine animals were subcutaneously transplanted into the right abdomen in an amount of 1 × 10 ⁶ [hereinafter referred to as IL-18 group]. As a control, Lewis lung cancer cells into which only the animal cell expression vector pRC / CMV was introduced were similarly subcutaneously transplanted in an amount of 1 × 10 ⁶ cells (hereinafter referred to as a control group). Up to 4 days after transplantation, no tumor engraftment was observed in both the control group and the IL-18 group. On day 7 and 11 days after implantation after transplantation, the tumor diameter was measured, the estimated tumor weight ^{formula: L × (W) 2/} 2 ( where, L is the length of the tumor, W is the tumor Width)
Was calculated. The results are as shown in Table 1 below.

Table 1 Estimated tumor weight on day 7 Estimated tumor weight on day 11 0.25 ± 0.14 g 1.03 ± 0.49 g IL-18 group 0.08 ± 0.02 g 0.20 ± 0 .10g

On day 17 after transplantation, two of the mice in the control group died due to the growth of the tumor, but IL-1
All eight groups were alive. On the 19th day after transplantation, the mice were sacrificed by cervical dislocation, the tumors were excised, and the final tumor weight was measured. Table 2 shows the results.

[Table 2] As described above, a significant tumor growth inhibitory effect was observed in the IL-18 group. In addition, when the tumor was observed with the naked eye, in some control group mice, growth to the outside of the epidermis was observed, but in the IL-18 group mouse,
No such growth outside the epidermis was observed. Furthermore, there was no difference in spleen size between the two groups, and there was no apparent metastatic lesion in the abdominal cavity or pleural cavity in both groups.

[0028]

The antitumor agent according to the present invention contains the gene itself, and by administering the antitumor agent particularly locally, tumors (especially solid cancers) can be reduced very effectively.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Eiji Inoguchi 734-2 Kita-Narushima-cho, Tatebayashi-shi, Gunma (72) Inventor Keiko Ishioka 2-180-8 Suzukicho, Kodaira-shi, Tokyo No.8 Building 103

Claims (1)

[Claims] 1. An interleukin comprising a gene encoding interleukin-18, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of interleukin-18. An antitumor agent comprising a complex of a gene encoding a protein having -18 activity and (2) a liposome.