WO2016190387A1 - Therapeutic agent for tumors, and use thereof - Google Patents

Abstract

A therapeutic agent for tumors according to the present invention contains apoptosis inhibitor of macrophage (AIM) or a protein variant having substantially the same activity as that of the AIM as an active ingredient and contains substantially no complement.

Description

Tumor therapeutic agent and use thereof

The present invention relates to a therapeutic agent for a tumor, a pharmaceutical composition for treating a tumor, a method for treating a tumor, and an AIM for producing the therapeutic agent for a tumor or a protein variant having substantially the same activity as the AIM. Regarding use.
This application claims priority based on provisional application No. 62 / 167,274 filed in the United States on May 28, 2015, the contents of which are incorporated herein by reference.

Non-epithelial cell-derived malignant tumors (so-called sarcomas) are malignant tumors derived from mesoderm that occur in bones, soft tissues, blood vessels, blood systems, and the like. What can be removed is eligible for surgery, but chemotherapy is a common treatment if metastases are advanced, have multiple manifestations, or are blood-borne tumors . Various anticancer agents have been researched and developed so far, and progress has been made, but conventional anticancer agents have strong side effects and may be difficult to use. On the other hand, although chemotherapy with molecular targeted drugs can exert a specific and powerful effect, it is effective for only a small part of cancer cells in which the target molecule is expressed. Development of molecular target drugs is desired.

Apotosis Inhibitor of Macrophage (AIM) is a protein with a molecular weight of about 40 k that is specifically expressed in macrophages. AIM was discovered as an apoptosis inhibitor and is known to have an anticancer effect via complement in hepatocellular carcinoma (see, for example, Non-Patent Document 1).

Non-Patent Document 1 discloses that accumulation of AIM on the cell surface in hepatocellular carcinoma activates complement and causes cytotoxicity to cause cancer cell-specific necrosis. There is little follow-up on AIM.

The present invention has been made in view of the above circumstances, and provides a novel therapeutic agent for tumors that is highly effective against various malignant tumors.

As a result of intensive studies to achieve the above object, the present inventors have found that AIM has a cell-killing effect on various malignant tumors by a mechanism not involving complement, and has completed the present invention. It was.

The present invention includes the following aspects.
[1] A therapeutic agent for tumors comprising an apoptosis inhibitor of macrophage (AIM) or a protein variant having substantially the same activity as that of the AIM as an active ingredient and substantially no complement.
[2] Contains an amino acid sequence that is 60% or more identical to the amino acid sequence shown in SEQ ID NO: 1 and has a cytocidal effect on tumor cells, a derivative thereof, or a salt or ester thereof as an active ingredient The therapeutic agent according to [1].
[3] The therapeutic agent according to [1] or [2], wherein the tumor is sarcoma (sarcoma).
[4] A pharmaceutical composition for treating a tumor, comprising the therapeutic agent according to any one of [1] to [3] and a pharmaceutically acceptable carrier or diluent.
[5] A method for treating a tumor, comprising administering to a mammal an effective amount of AIM or a protein variant having substantially the same activity as the AIM.
[6] The method for treating a tumor according to [5], wherein the mammal is a non-human mammal.
[7] The method for treating a tumor according to [6], wherein the non-human mammal is a dog.
[8] Use of AIM or a protein variant having substantially the same activity as AIM for producing a therapeutic agent for tumors.

According to the present invention, it is possible to provide a therapeutic agent for tumors that is highly effective against various malignant tumors.

It is a graph which shows the cell survival rate in the various tumor cells which added AIM of each concentration in Example 1. 2 is a photograph showing the presence or absence of apoptosis by genomic DNA electrophoresis in a histiocytic sarcoma cell line (CHS-5) with or without AIM in Example 1. FIG.

<< Tumor therapeutic agent >>
In one embodiment, the present invention comprises an apoptosis inhibitor of macrophage (AIM) or a protein variant having substantially the same activity as the AIM as an active ingredient, and a therapeutic agent for tumors substantially free of complement. I will provide a.

According to the tumor therapeutic agent of this embodiment, various malignant tumors can be effectively treated.

In the present specification, “containing as an active ingredient” means containing a therapeutically effective amount of a peptide.
In the present specification, “substantially free of complement” means that it contains no complement at all, or contains only a trace amount that does not activate and cause cell damage.
Therefore, in the tumor therapeutic agent of this embodiment, AIM or a protein variant having substantially the same activity as AIM has a cytocidal effect on tumor cells by a new mechanism not involving complement. .

In general, “Apoptosis Inhibitor of Macrophage (AIM)” is a protein with a molecular weight of 40k that is specifically produced by differentiated and matured macrophages, and is known to consist of three scavenger receptor cysteine-rich (SRCR) domains. It has been.

The AIM used in this embodiment may be derived from a mammal, and examples of the mammal include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, A goat, a marmoset, a monkey, a human, etc. are mentioned, It is not limited to these.

A protein variant having substantially the same activity as AIM has, for example, 60% or more identity with the amino acid sequence of AIM, preferably 70% or more, and more preferably 80% or more. More preferably, the protein is 90% or more, particularly preferably 95% or more.

A protein variant having substantially the same activity as AIM is, for example, a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of AIM. Good.
Here, the number of amino acids that may be deleted, substituted, or added is preferably 1 to 15, more preferably 1 to 10, and even more preferably 1 to 5.

Furthermore, the protein variant has substantially the same activity as AIM, that is, has a cytocidal effect on tumor cells.
In the present specification, the “cytocidal effect on tumor cells” means an effect of specifically killing tumor cells, for example, by promoting apoptosis.

In this specification, “tumor” mainly indicates a malignant tumor (cancer), and infiltrates surrounding tissues in a cell population (tumor) that has grown autonomously and uncontrolled by gene mutation. Or a malignant neoplasm that causes metastasis.
In the present specification, “cancer” is used to represent a diagnosis name, and “cancer” is used to represent a generic name of malignant neoplasm.
In general, the type of tumor is differentiated by the cells that develop, "solid cancer", where tumor cells gather to form isolated lesions, and "humoral", where tumor cells develop mainly in the blood due to abnormal hematopoietic tissue It is roughly divided into “cancer”. Furthermore, “solid cancer” can be divided into malignant tumors derived from epithelial cells and other malignant tumors derived from non-epithelial cells.

More specifically, examples of the solid cancer include breast tumors (eg, invasive ductal cancer, non-invasive ductal cancer, inflammatory breast cancer, etc.), prostate cancer, gastrointestinal tumors (eg, gastrointestinal stromal) Tumor, gastrointestinal adenocarcinoma), lung cancer (eg lung adenocarcinoma, lung squamous cell carcinoma, malignant mesothelioma), lymphoma (eg cutaneous lymphoma, intranasal lymphoma, digestive lymphoma, multicentric lymphoma) , Liver cancer (eg, hepatocellular carcinoma, hepatic carcinoid, extrahepatic bile duct cancer), kidney cancer (eg, renal cell carcinoma, nephroblastoma), gallbladder cancer, bile duct cancer, pancreatic cancer (eg, insulinoma) , Glucagonoma), ovarian tumor, uterine leiomyosarcoma, bladder cancer (eg transitional cell carcinoma), skin cancer (eg squamous cell carcinoma, malignant melanoma, cutaneous histiocytoma), thyroid cancer, hemangiosarcoma, blood vessel Fibrosarcoma, perianal adenocarcinoma, anal sac apocrine adenocarcinoma, testis Tumors (eg, Sertoli cell tumor, spermatoma, Leydig cell tumor), schwannomas, brain tumors (eg, meningiomas, gliomas), adrenal tumors, pituitary adenocarcinomas, osteosarcoma, cervical adenocarcinoma, obesity Celluloma, histiocytic sarcoma (for example, Langerhans cell histiocytosis, hemophagocytic histiocytosis) and the like are included, but are not limited thereto.

More specifically, the liquid cancer includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), malignant lymphoma, multiple myeloma, and the like. It is not limited to these.

The tumor to which the therapeutic agent for tumor of this embodiment is applied is not particularly limited as long as it is listed above, but among them, the therapeutic agent for tumor of this embodiment is for sarcoma. Are preferably used.

SEQ ID NO: 1 is the amino acid sequence of AIM in dogs.
More specifically, as a protein variant having substantially the same activity as AIM, for example, the identity with the amino acid sequence shown in SEQ ID NO: 1 is 60% or more (preferably 70% or more, more preferably 80% or more, More preferably 90% or more, particularly preferably 95% or more) and having a cytocidal effect on tumor cells, derivatives thereof, or salts or esters thereof as active ingredients, etc. Is mentioned.

The AIM or a protein variant having substantially the same activity as the AIM may be composed of L-amino acids, D-amino acids, or a combination thereof. L-amino acids are naturally occurring amino acids, and D-amino acids are those in which the chirality of L-amino acid residues is reversed. It may also be chemically modified to increase the killing effect on the tumor cells or to optimize other physical properties.
That is, the therapeutic agent for tumors of the present embodiment has the activity substantially equivalent to the AIM or the AIM together with or instead of the AIM or a protein variant having an activity substantially equivalent to the AIM. The derivative | guide_body of the protein variant which has this may be included.

In addition, since the therapeutic agent for tumors of the present embodiment exerts a desired effect as long as it comes into contact with the cell membrane, it does not have to be permeable to cells.

The tumor therapeutic agent of the present embodiment has the AIM or a protein variant having substantially the same activity as the AIM, and / or a derivative of the AIM, or an activity substantially equivalent to the AIM. In addition to or instead of a derivative of a protein variant, a salt of the AIM, or a salt or ester of a protein variant having substantially the same activity as the AIM, and / or a derivative of the AIM, or the A salt or ester of a derivative of a protein variant having substantially the same activity as AIM may be included.

The salt is preferably a pharmaceutically physiologically acceptable acid addition salt or basic salt.
Acid addition salts include, for example, salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid; acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid And salts with organic acids such as benzoic acid, methanesulfonic acid, and benzenesulfonic acid.
Examples of the basic salt include salts with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and magnesium hydroxide; salts with organic bases such as caffeine, piperidine, trimethylamine and pyridine. .
As the ester, for example, a conventional pharmaceutically acceptable one in the carboxyl group in the case of having a carboxyl group is preferable. Specifically, methyl group, ethyl group, propyl group, isopropyl group, butyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, etc. Esters with lower alkyl groups; esters with lower alkyl groups such as allyl groups and 2-butenyl groups; esters with lower alkoxy lower alkyl groups such as methoxymethyl groups, 2-methoxyethyl groups and 2-ethoxyethyl groups Can be mentioned. Here, the lower alkyl group means an alkyl group having 1 to 6 carbon atoms.

The tumor therapeutic agent of the present embodiment may contain, as other components, for example, a buffer solution such as PBS or Tris-HCl; an additive such as sodium azide or glycerol.

In the present embodiment, a method for treating a tumor can be provided by using the above-described tumor therapeutic agent. Examples of the tumor include those similar to those exemplified above.
The subject of treatment is not limited, and includes mammals including human or non-human animals. Examples of mammals include the same as those exemplified above.

<Method for producing tumor therapeutic agent>
The AIM used in the tumor therapeutic agent of the present embodiment may be naturally derived, or may be obtained by artificial chemical synthesis.
AIM derived from a natural product may be obtained directly from a body fluid sample (eg, blood sample), tissue, or cell derived from a non-human mammal by known recovery and purification methods, or known genetic recombination. Depending on the technique, the gene encoding the peptide may be incorporated into various expression vectors or the like, introduced into cells and expressed, and then obtained by known recovery and purification methods. Alternatively, commercially available kits such as reagent kits PROTEIOSTM (manufactured by Toyobo), TNTTM System (manufactured by Promega), PG-MateTM (manufactured by Toyobo) and RTS (manufactured by Roche Diagnostics), etc. are used. The AIM may be produced by a conventional cell-free protein synthesis system and may be obtained by a known recovery method and purification method, and is not limited.
Moreover, AIM by chemical synthesis can be obtained using a known protein synthesis method. Examples of the synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, and a redox method. In addition, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. A commercially available protein synthesizer may be used. After the synthesis reaction, AIM can be purified by combining known purification methods such as chromatography.

More specifically, for example, the AIM synthesis method can be prepared by the following method.
First, a host is transformed with an expression vector containing a nucleic acid encoding AIM. Subsequently, the host is cultured to express AIM. Conditions such as medium composition, culture temperature, time, addition of inducer, etc. can be determined by those skilled in the art according to known methods so that transformants grow and AIM is efficiently produced. For example, when an antibiotic resistance gene is incorporated into an expression vector as a selection marker, a transformant can be selected by adding an antibiotic to the medium. Subsequently, AIM is obtained by purifying AIM expressed by the host by an appropriate method.

<Method of administering tumor therapeutic agent>
The dosage of the tumor therapeutic agent of this embodiment takes into account the age, sex, weight, symptoms, treatment method, administration method, treatment time, etc. of the test animal (various mammals including human or non-human animals). Adjust as appropriate.
For example, when AIM or a protein variant having substantially the same activity as that of AIM is intravenously injected with an injection, the local concentration of AIM in a single administration is determined by AIM in a healthy subject. It is preferable to administer the blood concentration to be twice or more of the blood concentration, that is, 200 μM or more, more preferably 200 μM or more and 200 mM or less, and more preferably 2 mM or more and 20 mM or less. Particularly preferred.

Moreover, the tumor therapeutic agent of this embodiment can confirm the death of tumor cells 30 minutes after administration, preferably 4 hours to 48 hours, more preferably 8 hours to 30 hours, Preferably, 95% or more of tumor cells can be killed in 12 hours or more and 24 hours or less. The tumor therapeutic agent of this embodiment can kill tumor cells more rapidly than conventional anticancer agents, and further, this rapid cell killing effect does not depend on the mechanism through conventional complement. This is probably due to a new mechanism.

The number of administration is preferably 1 to several times per week.
Examples of the dosage form include intraarterial injection, intravenous injection, subcutaneous injection, intramuscular injection, eye drop, nasal drop, ear drop, intrabronchial administration, oral administration, or transdermal absorption, among others. Intravenous injection is preferred.
Injectables can also be prepared as non-aqueous diluents (eg, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol), suspensions, or emulsions. Such sterilization of injections can be performed by blending filter sterilization with a filter, bactericides, and the like. Injectables can be manufactured in the form of business preparation. That is, it can be used as a sterile solid composition by lyophilization, etc., and dissolved in distilled water for injection or other solvent before use.

<< Pharmaceutical composition for treatment of tumor >>
The pharmaceutical composition for treating a tumor of this embodiment comprises a therapeutically effective amount of the above-mentioned tumor therapeutic agent and a pharmaceutically acceptable carrier or diluent.

Pharmaceutically acceptable carriers or diluents include excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners, flavoring agents. Agents, solubilizers, additives and the like. By using one or more of these carriers, pharmaceutical compositions in the form of injections, solutions, capsules, suspensions, emulsions, syrups and the like can be prepared.

Also, a colloidal dispersion system can be used as the carrier. The colloidal dispersion system is expected to have an effect of enhancing the in vivo stability of the peptide and an effect of enhancing the transferability of the peptide to a specific organ, tissue, or cell. Examples of colloidal dispersion systems include polyethylene glycol, polymer composites, polymer aggregates, nanocapsules, microspheres, beads, oil-in-water emulsifiers, micelles, mixed micelles, and lipids including liposomes. Liposomes such as liposomes and artificial membranes that are effective in efficiently transporting peptides to specific organs, tissues, or cells are preferred.

Examples of the formulation in the pharmaceutical composition for treatment of tumor of this embodiment include those used orally as tablets, capsules, elixirs, and microcapsules with sugar coating as necessary.
Or what is used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension. Furthermore, a pharmacologically acceptable carrier or diluent, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, preservative , And those formulated by mixing in a unit dosage form generally required for pharmaceutical practice, in appropriate combination with a binder and the like.

Additives that can be mixed into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, swelling such as corn starch, gelatin, and alginic acid Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry. When the dispensing unit form is a capsule, the above material can further contain a liquid carrier such as fats and oils. Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.

Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride. Suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50 may be used in combination.

Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

<< Method of using tumor therapeutic agent and pharmaceutical composition for tumor treatment >>
One aspect of the present invention also provides the above-described AIM for treating tumors, or protein variants having substantially the same activity as the AIM, derivatives thereof, or salts or esters thereof.
One aspect of the present invention also provides a therapeutically effective amount of the above-mentioned AIM, or a protein variant having substantially the same activity as the AIM, a derivative thereof, or a salt or ester thereof, and pharmaceutically Pharmaceutical compositions comprising an acceptable carrier or diluent are provided.
Moreover, one aspect of the present invention provides a therapeutic agent for tumors, which comprises the above-described pharmaceutical composition for treating tumors.

Another aspect of the present invention is the use of the above-described AIM, or a protein variant having substantially the same activity as the AIM, a derivative thereof, or a salt or ester thereof for producing a therapeutic agent for tumors I will provide a.

Another aspect of the present invention is a patient in need of treatment with an effective amount of the above-mentioned AIM, or a protein variant having substantially the same activity as the AIM, a derivative thereof, or a salt or ester thereof. A method of treating a tumor comprising administering to a tumor.

Examples of the tumor to which the above-described treatment method is applied include those similar to those exemplified in the above << Tumor therapeutic agent >>.
In addition, examples of animals to which the above-described treatment methods are applied include humans and non-human mammals. Non-human mammals include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, marmosets, monkeys, and the like. Among them, the non-human mammal to which the above-described treatment method is applied is preferably a dog.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples and the like.

[Example 1]
(1) Preparation of recombinant canine AIM (1-1) Production of HA-tagged canine AIM expression vector SEQ ID NO: 2 is the nucleotide sequence of a nucleic acid encoding canine AIM. By using the nucleic acid encoding canine AIM as a template and PCR using AmpliTaq Gold (Applied Biosystems) using the primers shown in Table 1, the restriction enzyme sites of NotI and XhoI were respectively added to the 5 ′ end and the 3 ′ end. PCR product was obtained. Subsequently, a restriction enzyme treatment with NotI and XhoI was performed on the pCAGGS vector (pCAGGS-Leader Sequence-HNX-HA), and a nucleic acid (SEQ ID NO: 2) encoding canine AIM was inserted to prepare a canine AIM expression vector. The produced vector contains a cytomegalovirus enhancer, chicken β-actin promoter, rabbit β-globulin exon and intron, nucleic acid encoding HA polypeptide, and nucleic acid encoding canine AIM.

(1-2) Transfection of HA-tagged canine AIM expression vector into canine histiocytic sarcoma cell line CHS-5 Next, the canine histiocytic sarcoma cell line CHS-5 was transferred to a 1.0 mm × 10 ⁶ dish in a 90 mm dish. Cells were seeded so that 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 μg / mL streptomycin-containing DMEM (Dulbecco's Modified Eagle Medium) was added and cultured at 37 ° C. for 24 hours. did. The cultured CHS-5 cells were then transfected with a HA-tagged canine AIM expression vector at 1.25 μg / mL and cultured at 37 ° C. for 48 hours.

(1-3) Purification of HA-tagged dog AIM Next, the transfected cells were washed with PBS (−), and 1 mL of Lysis buffer (0.5 mL of 1 M Tris-HCl (pH 7.4), 0.1 mL) Of 1.5 M NaCl and 1 μL Triton-X 100 containing 0.4 mL ultrapure water). Next, the HA-tagged dog AIM was purified using Anti-HA Affinity Matrix (Roche). The eluate was prepared so that 50 mM glycine, 100 mM NaCl, 0.5% Tween was dissolved in 100 mL of ultrapure water to pH 2.0. The system was replaced with PBS at 4 ° C. for 24 hours. Subsequently, it concentrated by the ultrafiltration using Centriprep 30kDa (made by Merck Millipore), and dog AIM with a HA tag was obtained. The concentration of the obtained HA-tagged dog AIM was measured using NanoDrop Lite (manufactured by Thermo Fisher Scientific) and found to be 0.7 mg / mL. The obtained HA-tagged dog AIM was subjected to SDS-PAGE and confirmed by CBB staining and Western blotting using an anti-HA antibody (not shown).

(2) Verification of cell killing effect by recombinant AIM in various tumor cells Five types of canine histiocytic sarcoma cell lines (CHS-4, CHS-5, CHS-7, MHT-2, DH82) and 5 Other types of tumor cell lines (2 types of malignant melanoma (CMM1, CMM11), 2 types of mammary tumors (CTBp, CHMp), and transitional cell carcinoma (TCCUB)) were each added to a 96-well plate at 1.0 × 10 ^4. The cells were seeded to form cells, and 100 μL of DMEM used in (1-2) of Example 1 was added, followed by culturing at 37 ° C. for 24 hours under 5% CO ₂ . Then, 0M, 1.58 × 10 ⁻⁹ M (5.85 ng / 100 μL DMEM), 5 × 10 ⁻⁹ M (18.5 ng / 100 μL DMEM), 1.58 × 10 ⁻⁸ M (58.5 ng / 100 μL) (DMEM), 5 × 10 ⁻⁸ M (185 ng / 100 μL DMEM), 1.58 × 10 ⁻⁷ M (585 ng / 100 μL DMEM), 5 × 10 ⁻⁷ M (1.85 μg / 100 μL DMEM) The medium was replaced with DMEM containing the recombinant AIM prepared in 1) and cultured for 24 hours. Subsequently, the number of cells after the culture was counted using Cell Counting Kit-8 (manufactured by Dojindo). The cell viability at each concentration of recombinant AIM was calculated by setting the number of cells without recombinant AIM (0 M) in each cell type as 100%. The results are shown in FIG. In FIG. 1, the vertical axis indicates the number of viable cells (%), and the horizontal axis indicates the concentration of recombinant AIM.

From FIG. 1, it was clarified that the number of cells decreased in all 5 types of canine histiocytic sarcoma cell lines depending on the concentration of recombinant AIM. It was also revealed that the number of cells in CMM11, which is a malignant melanoma, and CHMp, which is a breast tumor, decrease depending on the concentration of recombinant AIM. In addition, in CMM1, which is a malignant melanoma, it has been revealed that the number of cells is significantly reduced at a concentration of recombinant AIM of 1 × 10 ⁻⁷ M or more.
From the above, it has been clarified that recombinant AIM has a cell-killing effect on many types of tumor cells, and particularly has a remarkable cell-killing effect on sarcoma (sarcoma).

(3) Verification of apoptosis-promoting action by AIM in histiocytic sarcoma cell line (CHS-5) The canine histiocytic sarcoma cell line CHS-5 was adjusted to 2.0 × 10 ⁵ cells / well in a 6-well plate. After seeding, 2 mL of DMEM used in (1-2) of Example 1 was added, and the mixture was cultured at 37 ° C. for 24 hours. Subsequently, the medium was replaced with DMEM containing recombinant AIM prepared in (1) of 3.2 × 10 ⁻⁷ M (1.2 μg / 100 μL DMEM), and cultured at 37 ° C. for 30 minutes. As a negative control, a recombinant AIM-free addition was prepared, and as a positive control, 10 ng / mL vincristine sulfate (manufactured by Sigma) was prepared. Subsequently, after washing with PBS, the cells were recovered with 200 μL of cell lysate. Next, 4 μL of RNase A (20 mg / mL, manufactured by Qiagen) and 4 μL of proteinase K (20 mg / mL, manufactured by Qiagen) were added to the obtained cell lysate and allowed to stand at 50 ° C. for 30 minutes. Next, 20 μL of 125 mM EDTA, 20 μL of 3M acetic acid, and 480 μL of isopropyl alcohol were added and mixed well. Subsequently, after centrifugation at 14,000 rpm for 15 minutes at 4 ° C., the sediment was dissolved in 15 μL of ultrapure water. Subsequently, the obtained DNA solution was developed by electrophoresis on a 2.0% TBE agarose gel. After running at 100 V for about 40 minutes, DNA was stained with ethidium bromide. The results are shown in FIG. In FIG. 2, M represents a marker, P represents a positive control, N represents a negative control, and A represents an AIM addition group.

FIG. 2 qualitatively shows that DNA fragmentation (apoptosis) significantly increases in the recombinant AIM addition group.
In addition, remarkable DNA fragmentation was observed 30 minutes after the addition of recombinant AIM, suggesting that apoptosis was rapidly induced as compared with conventional anticancer agents.

[Example 2]
(1) Preparation of recombinant canine AIM Using the same method as in (1) of Example 1 except that HeLa cells were used instead of the canine histiocytic sarcoma cell line CHS-5 as cells to be transfected, HA-tagged dog AIM was prepared.

(2) Verification of cell killing effect by recombinant AIM in various tumor cells The cell types shown in Table 2 below were suspended in 1 mL of DMEM used in (1-2) of Example 1 above, It seed | inoculated so that it might become 1.0 * 10 < ⁵ > cells / well to a 24-well plate, and it culture | cultivated at 37 degreeC for 24 hours. Subsequently, the recombinant AIM prepared in (1) was added to 1 × 10 ⁻⁷ M and cultured at 37 ° C. for 48 hours.
Next, the cultured cells were washed twice with PBS, and the number of viable cells and dead cells were measured using AOPI (manufactured by Nexus Bioscience), and the cell viability was calculated. The results are shown in Table 2. In Table 2, sensitivity is the result of evaluating other AIM sensitivities when the AIM sensitivity in DH82 and HeLa cells is moderate. Compared with DH82 and HeLa cells, cells with high cell viability have low AIM sensitivity, cells with similar cell viability have medium AIM sensitivity, and cells with low cell viability have AIM sensitivity. Was high.

From Table 2, the osteosarcoma D17, the mammary gland osteosarcoma MCO-Y4, the fibrosarcoma FC81. In T25, which is erythroleukemia, a remarkable cell killing effect was observed. In addition, F1B, which is a lymphoma, showed a cytocidal effect similar to that of DH82 and HeLa.
Therefore, it has been clarified that recombinant AIM has a cell-killing effect on all types of tumor cells, and particularly has a remarkable cell-killing effect on sarcoma (sarcoma).

According to the therapeutic agent of the present invention, various malignant tumors can be effectively treated.

Claims (8)

1. A therapeutic agent for tumors comprising, as an active ingredient, Apoptosis Inhibitor of Macrophage (AIM) or a protein variant having substantially the same activity as AIM as an active ingredient.
2. A protein, derivative thereof, or a salt or ester thereof containing an amino acid sequence having an identity of 60% or more with the amino acid sequence shown in SEQ ID NO: 1 and having a cytocidal effect on tumor cells, as an active ingredient 1. The therapeutic agent according to 1.
3. The therapeutic agent according to claim 1 or 2, wherein the tumor is sarcoma.
4. A pharmaceutical composition for treating a tumor, comprising the therapeutic agent according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or diluent.
5. A method for treating a tumor, comprising administering an effective amount of AIM or a protein variant having substantially the same activity as the AIM to a mammal.
6. The method of treating a tumor according to claim 5, wherein the mammal is a non-human mammal.
7. The method for treating a tumor according to claim 6, wherein the non-human mammal is a dog.
8. Use of AIM or a protein variant having substantially the same activity as AIM for producing a therapeutic agent for tumors.

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