JP2002326949A - Immunostimulator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To treat cancers and virus infectious diseases by stimulating the immune system. SOLUTION: This immunostimulator contains a TGF-γ receptor or its derivative, and an MHC binding peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an immunostimulant and an anticancer agent and an antiviral agent which act by activating the immune system.

[0002]

BACKGROUND ART Cancer-associated antigen (TAA) peptides have been discovered, and specific cytotoxic T cells (CTL) have been experimentally induced. However, cancer-bearing animals have not been able to completely reject cancer cells.

[0003] An object of the present invention is to provide an anticancer agent which can strongly activate cellular immunity and completely reject cancer cells.

Further, the present invention strongly activates cellular immunity,
It is an object of the present invention to provide a drug for treating viral infection by attacking cells infected with a virus such as HIV.

[0005]

Means for Solving the Problems The present inventors have considered that TGF-secreted by cancer cells is a factor in which CTL is not activated effectively.
We thought that it was possible to completely eliminate cancer cells by administering a composition containing a TGF-β receptor and an MHC-binding peptide to a tumor-bearing animal, which was thought to suppress immunocompetent cells by β. Was.

The present invention relates to the following items 1 to 5. Item 1. An immunostimulant comprising a TGF-β receptor or a derivative thereof and an MHC-binding peptide. Item 2. MHC-binding peptide is MAGE-1 (Glu Ala As
p Pro Thr Gly His SerTyr), MART-1 (Ala Ala Gly Il
e Gly Ile Leu Thr Val), MUM-1 (Glu Glu LysLeu Ile
Val Val Leu Phe), Ovalbumin OVA 257-264 (Ser
Ile Ile Asn Phe Glu Lys Leu), Influenza NP265-
273 (Ile Leu Arg Gly Ser Val Ala HisLys), HIVgp41
583-591 (Arg Tyr Leu Lys Asp Gln Leu Leu) and HIV
p24 gag267-275 (Ile Val Gly Leu Asn Lys Ile Val Ar
Item 1 which is at least one member selected from the group consisting of g)
3. The immunostimulant according to item 1. Item 3. The immunostimulator according to claim 1 or 2, wherein the TGF-β receptor or a derivative thereof contains an extracellular portion of TGF-βRII. Item 4. Item 4. The immunostimulant according to any one of Items 1 to 3, which is an anticancer agent. Item 5. Item 4. The immunostimulant according to any one of Items 1 to 3, which is an antiviral agent.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the MHC-binding peptide is not particularly limited as long as it is a peptide capable of binding to MHC (major histocompatibility complex), but the peptide preferably has 7 to 12 amino acids. , More preferably 8 to 10. Specific examples of such MHC-binding peptides include MAGE-1 (Glu Ala Asp Pro
Thr Gly His Ser Tyr), MART-1 (Ala Ala Gly Ile Gly
Ile Leu Thr Val) and MUM-1 (Glu Glu Lys Leu Ile V
alval Leu Phe) and other peptides derived from malignant melanoma, ovalbumin (OVA; 257-264): Ser Ile Ile Asn Phe G
lu Lys Leu, Influenza NP265-273 (Ile Leu Arg G
ly Ser Val Ala His Lys), HIV gp41 583-591 (Arg Tyr
Leu Lys Asp Gln Leu Leu), HIVp24 gag 267-275 (Il
e Val Gly LeuAsn Lys Ile Val Arg). M
As long as HC binding is not lost, one or more peptides may be further bound to the N-terminus or C-terminus of the peptide. In addition, several hundred types of MHC-binding peptides other than the above-mentioned peptides have been identified, and any of these known peptides may be used.

[0008] The TGF-β receptor and its derivatives are used in mammals including humans (mouse, rat, rabbit, hamster, dog, monkey, pig, horse, cow, etc.), preferably human.
A TGF-β receptor, which is a portion exposed outside the cell (for example, in the case of a mouse, positions 24 to 1 of SEQ ID NO: 4)
59), and preferably does not include a cell membrane-binding portion or a portion present in a cell. The receptor may have one or more amino acids substituted, deleted or added as long as it binds to TGF-β. Preferred derivatives are polypeptides comprising the extracellular portion of the human TGF-β receptor.

Here, the TGF-β receptor includes TGF-βRII,
An example is secreted TGF-βRII (TGF-βRII Ig).

For example, mouse TGF-βRII has SEQ ID NO: 4
The amino acids at positions 1 to 23 are signal peptides, the extracellular portion is at positions 24 to 159, the transmembrane portion is at positions 160 to 189, and the portion after position 190 is the intracellular portion. .

[0011] SEQ ID NO: 5 contains mouse secreted TGF-
βRII is shown, positions 1 to 136 are the extracellular portion of mouse TGF-β receptor (identical to positions 24 to 159 of SEQ ID NO: 4)
And positions 137-372 are the Fc portion of human IgG1. The Fc portion has been linked to facilitate purification by affinity chromatography. A chimeric protein to which such a sequence is bound is also included in the TGF-β receptor of the present invention.

In the present invention, the mixing ratio of the TGF-β receptor or its derivative and the MHC-binding peptide is such that the MHC-binding peptide is added to 100 parts by weight of the TGF-β receptor.
It is about 0001 to 10 parts by weight.

When the pharmaceutical composition of the present invention is used as an immunostimulant for mammals including humans, in particular, a therapeutic agent (anticancer agent) for malignant tumors, various pharmaceutical administration forms can be widely used depending on the purpose. Specific examples of the form include oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions, injections, suppositories, ointments,
Parenteral preparations such as patches can be exemplified. These administration agents are formulated by conventional formulation methods generally known in the art.

In the case of molding into a tablet form, for example, excipients, binders, disintegrants, disintegration inhibitors, absorption promoters, humectants, adsorbents, lubricants and the like can be used as carriers. Further, the tablet can be a tablet coated with a usual coating, if necessary, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet or a multilayer tablet.

In the case of molding into a pill form, as a carrier, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, binders such as gum arabic powder, tragacanth powder, gelatin and the like; Laminaran,
Disintegrators such as agar can be used.

Capsules are usually prepared by mixing the compound of the present invention with the various carriers exemplified above and filling the mixture into hardened gelatin capsules, soft capsules, and the like.

When the suppository is formed, for example, polyethylene glycol, cocoa butter, higher alcohol, esters of higher alcohol, gelatin, semi-synthetic glyceride and the like can be used as carriers.

When prepared as an injection, the liquid preparation, emulsion and suspension are preferably sterilized and isotonic with blood. When formed into these forms, water, ethyl alcohol or the like may be used as a diluent. Macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and the like. In this case, a sufficient amount of salt, glucose or glycerin to prepare an isotonic solution may be included in the pharmaceutical preparation, and a usual solubilizing agent, a buffer, a soothing agent and the like may be added. You may.

When preparing an ointment, a base, a stabilizer, a wetting agent, a preservative and the like usually used for the compound of the present invention are blended if necessary, and mixed and formulated by a conventional method.

In the case of producing a patch, the above-mentioned ointment, paste, cream, gel or the like may be applied to a usual support in a conventional manner. As the support, woven cloth, non-woven cloth and soft vinyl chloride, polyethylene,
A film such as polyurethane or a foam sheet is suitable.

Further, in each of the above-mentioned preparations, a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals may be contained in the pharmaceutical preparation, if necessary.

The amount of the composition of the present invention (total of receptor and peptide) to be contained in the pharmaceutical preparation of the present invention is as follows:
It is not particularly limited and may be appropriately selected over a wide range, or may be usually 1 to 70% by weight in a pharmaceutical preparation.

The method of administration of the above pharmaceutical preparation is not particularly limited, and is appropriately determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are administered orally. The injection is administered intravenously, alone or as a mixture with a normal replenisher such as glucose or amino acid, and further administered intramuscularly, intradermally, subcutaneously or intraperitoneally as needed. Suppositories are administered rectally. The ointment is applied to skin, oral mucosa, and the like.

The amount of the composition of the present invention (receptor and peptide) to be incorporated into each of the above dosage unit forms is not fixed depending on the condition of the patient to which the composition is to be applied or the dosage form thereof. About 0.0 for oral preparation per unit form
Desirably, the dose is from 0.01 to 100 mg, and about 0.01 to 50 mg for an injection. In addition, the daily dose of the drug having the above-mentioned administration form varies depending on the patient's condition, body weight, age, sex and the like and cannot be unconditionally determined, but is usually about 0.1 to 5000 mg, preferably 1 to 1000 mg per adult day.
g, which is preferably administered once or divided into about 2 to 4 doses.

The malignant tumor which can be treated by administering a preparation containing the compound of the present invention is not particularly limited, and examples thereof include head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer,
Liver cancer, gallbladder / bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, malignant lymphoma, leukemia, cervical cancer, skin cancer, brain tumor, etc. .

[0026]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in more detail with reference to embodiments.

The OVA of SEQ ID NO: 6 (257-264)
Was produced by an automatic peptide synthesizer.

The production conditions for OVA-liposomes are described in the literature (Proc. Natl. Acad. Sci. USA 89, 8995-8999, 199).
It is described in 2).

Further, the secreted TGF-
TGF-βRII Ig, βRII, has been described in the literature (Cancer Res. 59, 1).
273-1277, 1999). Example 1: Tumor rejection experiment of EG-7 EG-7 (10 * 6 / head; OVA gene-introduced tumor cells were injected subcutaneously into the back of B6 mice (8 weeks old, female, 5 animals per group)) ) To form a tumor having a diameter of about 8 mm.

The mouse carrying the tumor was subjected to (1) OVA
Liposomes (100 μg / head) alone, (2) OVA
-Liposome (100 µg / head) + TGF-βRII Ig (2
(3) TGF-βRII Ig (200 μg / head)
head) alone was inoculated subcutaneously near the tumor after tumor formation.

FIG. 1 shows the results. In the case of (1) OVA-liposome administration alone and (2) TGF-βRIIIg administration alone, all died 16 days after administration.
In the case of the combined administration of VA-liposome and TGF-βRII Ig, all the mice survived even after 28 days, and three out of five mice completely disappeared the tumor.

[0032]

According to the immunostimulant of the present invention, cell-mediated immunity can be strongly activated, and it is extremely useful as an anticancer agent. In addition, by activating the immune system, it is expected to be useful for treating viral infections such as influenza virus, HIV, HTML, and hepatitis virus.

[0033]

[Sequence List] SEQUENCE LISTING <110> KANSAI TLO Co., Ltd. <120> Immuno-stimulative agent <130> 11701JP <160> 9 <170> PatentIn Ver. 2.0 <210> 1 <211> 9 <212> PRT <213> MAGE-1 <400> 1 Glu Ala Asp Pro Thr Gly His Ser Tyr 1 5 9 <210> 2 <211> 9 <212> PRT <213> MART-1 <400> 2 Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 9 <210> 3 <211> 9 <212> PRT <213> MUM-1 <400> 3 Glu Glu Lys Leu Ile Val Val Leu Phe 1 5 9 <210> 4 <211> 567 <212 > PRT <213> mouse TGF-βRII <400> 4 Met Gly Arg Gly Leu Leu Arg Ala Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Pro Lys Ser Val 20 25 30 Asn Ser Asp Val Met Ala Ser Asp Asn Gly Gly Ala Val Lys Leu Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Leu Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ala Ile Cys Glu Lys Pro 65 70 75 80 His Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Lys Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Thr Tyr His Gly Phe Thr 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Val Met Lys Glu Lys Lys Arg 115 120 125 Ala Gly Glu Thr Phe Phe Met Cys Ala Cys Asn Met Glu Glu Cys Asn 130 135 140 Asp Tyr Ile Ile Phe Ser Glu Glu Tyr Thr Thr Ser Ser Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Ile Gln Val Thr Gly Val Ser Leu Leu Pro Pro Leu 165 170 175 Gly Ile Ala Ile Ala Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val His 180 185 190 Arg Gln Gln Lys Leu Ser Pro Ser Trp Glu Ser Ser Lys Pro Arg Lys 195 200 205 Leu Met Asp Phe Ser Asp Asn Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215 220 Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu 225 230 235 240 Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala 245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu 260 265 270 Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ser Ser Trp Lys 275 280 285 Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile 290 295 300 Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln 305 310 315 320 Tyr Trp Leu Ile ThrAla Phe His Arg Lys Gly Asn Leu Gln Glu Tyr 325 330 335 Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340 345 350 Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys 355 360 365 Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn 370 375 380 Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu 385 390 395 400 Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Lys Gln 405 410 415 Arg Glu Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser 420 425 430 Arg Met Asn Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440 445 Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Trp Asn Ala Val 450 455 460 Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu 465 470 475 480 His Pro Cys Val Glu Ser Met Lys Asp Ser Val Leu Arg Asp Arg Gly 485 490 495 Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Ile 500 505 510 Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg 515 520 525 Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Met 530 535 540 Asp Arg Leu Ser Gly Arg Ser Cys Ser Gln Glu Lys Ile Pro Glu Asp 545 550 555 560 Gly Ser Leu Asn Thr Thr Lys 565 567 <210> 5 <211> 372 <212> PRT <213> Secretion-type TGF-βRII <400> 5 Ile Pro Pro His Val Pro Lys Ser Val Asn Ser Asp Val Met Ala Ser 1 5 10 15 Asp Asn Gly Gly Ala Val Lys Leu Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Leu Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ala Ile Cys Glu Lys Pro His Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Lys Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Thr Tyr His Gly Phe Thr Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Val Met Lys Glu Lys Lys Arg Ala Gly Glu Thr Phe Phe Met 100 105 110 Cys Ala Cys Asn Met Glu Glu Cys Asn Asp Tyr Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Thr Thr Ser Ser Pro Asp Leu Asp Pro Glu Glu Pro Lys Ser 130 135 140 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 145 150 155 160 Gly Gly Pro Se r Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 165 170 175 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 180 185 190 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 195 200 205 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 210 215 220 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 225 230 235 240 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 245 250 255 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 260 265 270 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 275 280 285 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 290 295 300 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 305 310 315 320 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 325 330 335 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 340 345 350 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 355 360 365 Ser Pro Gly Lys 37 0 372 <210> 6 <211> 8 <212> PRT <213> OVA (257-264) <400> 6 Ser Ile Ile Asn Phe Glu Lys Leu 1 5 8 <210> 7 <211> 9 <212> PRT <213> Influenza NP265-273 <400> 7 Ile Leu Arg Gly Ser Val Ala His Lys 1 5 9 <210> 8 <211> 8 <212> PRT <213> HIVgp41 583-591 <400> 8 Arg Tyr Leu Lys Asp Gln Leu Leu 1 5 8 <210> 9 <211> 9 <212> PRT <213> HIVp24 gag 267-275 <400> 9 Ile Val Gly Leu Asn Lys Ile Val Arg 1 5 9

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the cancer reduction effect when OVA-liposome alone, (2) OVA-liposome + TGF-βRII Ig, and (3) TGF-βRII Ig alone are administered to tumor-bearing mice. The horizontal axis indicates days (day), and the vertical axis indicates tumor diameter (mm).

Claims (5)

[Claims] 1. An immunostimulator comprising a TGF-β receptor or a derivative thereof and an MHC-binding peptide. 2. The method according to claim 2, wherein the MHC-binding peptide is MAGE-1 (Glu Al
a Asp Pro Thr Gly His Ser Tyr), MART-1 (Ala Ala G
ly Ile Gly Ile Leu Thr Val), MUM-1 (Glu Glu Lys L
eu Ile Val Val Leu Phe), Ovalbumin OVA 257-26
4 (Ser Ile IleAsn Phe Glu Lys Leu), influenza N
P265-273 (Ile Leu Arg Gly Ser Val Ala His Lys), H
IVgp41 583-591 (Arg Tyr Leu Lys Asp Gln Leu Leu)
And HIVp24 gag 267-275 (Ile Val Gly Leu Asn Lys Il
The immunostimulator according to claim 1, which is at least one member selected from the group consisting of e Val Arg). 3. The method of claim 1, wherein the TGF-β receptor or a derivative thereof is TGF-βRI.
3. The immunostimulant according to claim 1, comprising the extracellular portion of I. 4. The immunostimulant according to claim 1, which is an anticancer agent. 5. The immunostimulant according to claim 1, which is an antiviral agent.