WO2013032028A1 - New compound material, medicine containing same, and cancer treatment method using same - Google Patents

Abstract

The present invention relates to a compound material of a gene transfer reagent and a vector of an encoded microbe-derived antigen protein gene, a medicine containing the same, and a cancer treatment method using the same.

Description

NOVEL COMPLEX, MEDIUM CONTAINING THE SAME AND CANCER TREATMENT METHOD

The present invention relates to a complex of a vector encoding a microorganism-derived antigen protein gene and a gene introduction reagent, a medicine containing them, and a method for treating cancer using them.

Gene therapy has been expected and attempted as a radical treatment method for treating a disease by giving a normal gene to a patient with a genetic disease caused by a gene deficiency or abnormality. The first gene therapy performed for therapeutic purposes following formal procedures was conducted in 1990 for patients with ADA (adenosine deaminase) deficiency. In order to introduce a DNA encoding a gene required for treatment into a target cell, a gene introduction system is often used. Currently, gene introduction systems include (1) a viral gene introduction system and (2) And non-viral gene transfer systems such as cationic polymers or cationic lipids.
Many gene therapy clinical trials that have been conducted so far utilize a viral gene transfer system. (1) Retrovirus (2) Adenovirus (Ad) (3) Adeno-associated virus (AAV) and the like have been used.
On the other hand, viruses that have been recombined to selectively kill only tumor cells using the cytolytic activity of the virus have been developed as a new technique for cancer gene therapy. These are called oncolytic viruses, restricted-proliferating viruses, and the like, and are proliferating in tumor cells, but are attracting attention as highly safe anti-tumor virus preparations because of their low proliferation activity in normal cells ( Non-patent literature ①).
Viruses that proliferate selectively in tumors have been found in nature, for example, by replacing the promoter of the E1 region of adenovirus with a promoter that is selectively expressed only in tumor cells. Can also be obtained. As tumor selective promoters, telomerase promoter, midkine promoter, IAI3B promoter and the like have been used.
On the other hand, from many experimental and clinical findings in recent years, the antitumor activity of these viruses is not only due to their own cytocidal action, but rather to induce antitumor immunity of the host, It is claimed that the effect to activate is great. That is, when an oncolytic virus is administered, anti-tumor immunity is elicited in the host, and the tumor effectively regresses due to its immune effect. This is supported by the regression of tumors at sites away from tumors treated with virus therapy and the resistance to re-transplantation of the same type of tumor cells that continues for a long time after treatment (Non-patent Document 2).
Viruses that are highly infectious to animal cells are used. However, many treatments require multiple administrations, and by repeating administration, antiviral antibodies are produced, and infection and gene transfer are significantly inhibited. Therefore, a treatment system using an oncolytic adenovirus or an adenovirus incorporating a p53 gene has been approved and used in China, but a sufficient healing effect has not been obtained. However, the non-viral gene transfer system has very low activity in vivo except for a few examples of the applicants (Non-patent Document 3), and has not led to a therapeutic effect.

Under such circumstances, the present inventors have examined in detail the mechanism of anti-tumor immune activation by oncolytic viruses, and as a result, the expression of microbial antigen protein in infected tumor cells or cells in the vicinity of the tumor is anti-tumor. We found the possibility of being involved in the establishment of tumor immunity. Tumor cells infected with viruses are thought to elicit these immune responses by presenting or secreting proteins unique to the microorganisms or their degradation products on the surface of the cell membrane.
The present inventors devised that expression of such microbial antigen protein in tumor cells can be achieved not only by viral infection but also by gene transfer using DNA. In addition, as a tool for gene transfer using DNA, we have a plasmid DNA system that is highly expressed in vivo as a unique technology (Non-patent Document 3). Therefore, plasmids encoding the genes of ESAT-6 and Ag85B, which are antigenic proteins derived from Mycobacterium tuberculosis, were constructed, a gene transfer system consisting of the ionic complex was prepared, and administered locally to tumor-bearing model mice. Then, a much higher antitumor effect was confirmed than the comparative example using the plasmid which coded the gene of GM-CSF which is a cytokine with a high antitumor effect.
Moreover, it is known that the simultaneous administration of GM-CSF is effective for the expression of the antitumor effect by the oncolytic virus. Therefore, when a plasmid complex encoding the ESAT-6 gene and a plasmid complex encoding the GM-CSF gene were mixed and administered intratumorally to a tumor-bearing model mouse, a plasmid encoding the ESAT-6 gene was obtained. A higher antitumor effect was observed than the complex alone. Alternatively, it has also been reported that a virus into which an immune-activating cytokine gene such as IL-2 or IL-12 has been introduced has high antitumor activity. Therefore, when a plasmid complex encoding a gene of ESAT-6 and a plasmid complex encoding a gene such as IL-2 and IL-12 were mixed and administered to a tumor-bearing model mouse locally, tumors of ESAT-6 were obtained. An antitumor effect higher than that of the plasmid complex encoding the gene alone was observed.

The present invention
(1) A complex containing a vector encoding a microorganism-derived antigen protein gene and a gene introduction reagent.
(2) The complex according to (1) above, wherein the microorganism is Mycobacterium tuberculosis.
(3) The complex according to (2), wherein the Mycobacterium tuberculosis-derived antigenic protein is ESAT-6 (M. tuberculosis 6 kDa early secreted antigenic target) and / or Ag85B.
(4) The complex according to any one of (1) to (3) above, wherein the vector is a plasmid vector.
(5) The complex according to any one of (1) to (4) above, wherein the gene introduction reagent comprises one or more compounds selected from a cationic polymer, a cationic lipid, and an anionic polymer.
(6) The complex according to (5) above, wherein the gene introduction reagent comprises a cationic polymer or a cationic lipid and an anionic polymer.
(7) The cationic polymer is polyethyleneimine (linear polyethyleneimine or polybranched ethyleneimine), 2-dimethylaminoethyl methacrylate polymer or copolymer, 2-trimethylaminoethyl methacrylate polymer or copolymer. Polymer, polyamidoamine dendrimer, polylysine dendrimer, protamine, histone, HelΔ1, gelatin, poly-L-lysine, polyarginine, polyornithine, polyvinylimidazole, a polymer having an ethylenediamine structure in the side chain and a copolymer thereof, or The complex according to (5) or (6) above, wherein at least one of these salts is selected.
(8) The cationic lipid is DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol, DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DMRI (N- (1,2-Dimyristyloxyppa-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- (2- (sperminecarboxamide) -N, N-dimethylammonium trifluoroacetate) ), DOTAP (N- (1- (2,3-dioleyloxy) propyl) -N, N, N-to Limethylammonium chloride) and DOTMA (N- (1- (2,3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride) and their salts with different acids The complex according to (5) or (6) above.
(9) PEG derivative having carboxyl side chain, polymer or copolymer of acrylic acid or methacrylic acid, sulfate ester of polyvinyl alcohol, succinimidylated poly-L-lysine, polyglutamic acid, polyaspartic acid , One or more of hyaluronic acid, chondroitin, chondroitin sulfate, keratan sulfate, heparin, deltaman sulfate or a derivative thereof, and a salt thereof.
(10) The composite according to any one of (1) to (9), which has been subjected to a freeze-drying treatment.
(11) A medicament containing a vector encoding a microorganism-derived antigen protein gene and a gene introduction reagent.
(12) The medicament according to (11) above, wherein the microorganism is Mycobacterium tuberculosis.
(13) The medicament according to (12) above, wherein the Mycobacterium tuberculosis-derived antigenic protein is ESAT-6 (M. tuberculosis 6 kDa early secreted antigenic target) and / or Ag85B.
(14) The medicament according to any one of (11) to (13) above, wherein the vector is a plasmid vector.
(15) The medicament according to any one of (11) to (14) above, wherein the gene introduction reagent comprises one or more compounds selected from a cationic polymer, a cationic lipid, and an anionic polymer.
(16) The medicament according to (15) above, wherein the gene introduction reagent comprises a cationic polymer or cationic lipid and an anionic polymer.
(17) The cationic polymer is polyethyleneimine (linear polyethyleneimine or polybranched ethyleneimine), 2-dimethylaminoethyl methacrylate polymer or copolymer, 2-trimethylaminoethyl methacrylate polymer or copolymer. Polymer, polyamidoamine dendrimer, polylysine dendrimer, protamine, histone, HelΔ1, gelatin, poly-L-lysine, polyarginine, polyornithine, polyvinylimidazole, a polymer having an ethylenediamine structure in the side chain and a copolymer thereof, or The medicament according to (15) or (16) above, wherein one or more salts thereof are selected.
(18) The cationic lipid is DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol, DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DMRI (N- (1,2-Dimyristyloxyppa-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- (2- (sperminecarboxamide) -N, N-dimethylammonium trifluoroacetate) ), DOTAP (N- (1- (2,3-dioleyloxy) propyl) -N, N, N- The trimethylammonium chloride) and DOTMA (N- (1- (2,3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride) and their salts with different acids. The medicine according to (15) or (16).
(19) PEG derivative having carboxyl side chain, polymer or copolymer of acrylic acid or methacrylic acid, sulfate of polyvinyl alcohol, succinimidylated poly-L-lysine, polyglutamic acid, polyaspartic acid The pharmaceutical according to any one of (15) to (18) above, which is selected from one or more of hyaluronic acid, chondroitin, chondroitin sulfate, keratan sulfate, heparin, deltaman sulfate or a derivative thereof, and salts thereof.
(20) The medicament according to any one of (11) to (19), which has been freeze-dried.
(21) A pharmaceutical composition comprising a vector encoding a microorganism-derived antigen protein gene and a complex containing a gene introduction reagent, and a combination of a vector encoding an immunostimulatory cytokine gene and a complex containing a gene introduction reagent.
(22) The medicament according to (21) above, wherein the microorganism is Mycobacterium tuberculosis.
(23) The medicament according to (22) above, wherein the Mycobacterium tuberculosis-derived antigenic protein is ESAT-6 (6 kDa early secreted antigenic target) and / or Ag85B.
(24) Any of the above (21) to (23), wherein the immunostimulatory cytokine is one or more cytokines selected from interleukin-2 (IL-2), interleukin-12 (IL-12), and GM-CSF The pharmaceutical described.
(25) A method for treating cancer, comprising administering an effective amount of a complex containing a vector encoding a microorganism-derived antigen protein gene and a gene introduction reagent.
(26) The method according to (25) above, wherein the microorganism is Mycobacterium tuberculosis.
(27) The method according to (26) above, wherein the Mycobacterium tuberculosis-derived antigenic protein is ESAT-6 (M. tuberculosis 6 kDa early secreted antigenic target) and / or Ag85B.
(28) The method according to any one of (25) to (27) above, wherein the vector is a plasmid vector.
(29) The treatment method according to any one of (25) to (28) above, wherein the gene introduction reagent comprises one or more compounds selected from a cationic polymer, a cationic lipid, and an anionic polymer.
(30) The treatment method according to the above (29), wherein the gene introduction reagent comprises a cationic polymer or a cationic lipid and an anionic polymer.
(31) The cationic polymer is polyethyleneimine (linear polyethyleneimine or polybranched ethyleneimine), 2-dimethylaminoethyl methacrylate polymer or copolymer, 2-trimethylaminoethyl methacrylate polymer or copolymer. Polymer, polyamidoamine dendrimer, polylysine dendrimer, protamine, histone, HelΔ1, gelatin, poly-L-lysine, polyarginine, polyornithine, polyvinylimidazole, a polymer having an ethylenediamine structure in the side chain and a copolymer thereof, or The treatment method according to the above (29) or (30), wherein one or more salts thereof are selected.
(32) The cationic lipid is DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol, DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DMRI (N- (1,2-Dimyristyloxyppa-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- (2- (sperminecarboxamide) -N, N-dimethylammonium trifluoroacetate) ), DOTAP (N- (1- (2,3-dioleyloxy) propyl) -N, N, N- The trimethylammonium chloride) and DOTMA (N- (1- (2,3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride) and their salts with different acids. (29) or the treatment method of (30) description.
(33) An anionic polymer is a PEG derivative having a carboxyl side chain, a polymer or copolymer of acrylic acid or methacrylic acid, a sulfate ester of polyvinyl alcohol, succinimidylated poly-L-lysine, polyglutamic acid, polyaspartic acid The method according to any one of (29) to (32) above, wherein at least one selected from hyaluronic acid, chondroitin, chondroitin sulfate, keratan sulfate, heparin, deltaman sulfate or a derivative thereof, and a salt thereof.
(34) The treatment method according to any one of (25) to (33) above, which has been lyophilized.
(35) An effective amount of a combination of a vector encoding a microorganism-derived antigen protein gene and a complex containing a gene introduction reagent and a vector encoding an immunostimulatory cytokine gene and a complex containing a gene introduction reagent are administered. A method for treating cancer.
(36) The method according to (35) above, wherein the microorganism is Mycobacterium tuberculosis.
(37) The method according to (36) above, wherein the Mycobacterium tuberculosis-derived antigenic protein is ESAT-6 (M. tuberculosis 6 kDa early secreted antigenic target) and / or Ag85B.
(38) Any of the above (35) to (37), wherein the immunostimulatory cytokine is one or more cytokines selected from interleukin-2 (IL-2), interleukin-12 (IL-12), and GM-CSF The method of treatment described.
(39) The complex or pharmaceutical according to any one of (1) to (7), (9) to (17), (19) to (31), and (33) to (37), wherein the cationic polymer is polyethyleneimine Alternatively, a method for treating cancer.
(40) The complex, pharmaceutical or cancer treatment method according to any one of (1) to (39), wherein the anionic polymer is chondroitin sulfate.
(41) The complex, pharmaceutical or cancer treatment method according to any one of (1) to (39), wherein the anionic polymer is hyaluronic acid.

The complex of the vector encoding the microorganism-derived antigen protein gene of the present invention and the gene introduction reagent is capable of efficiently introducing the M. tuberculosis-derived antigen protein gene into the cell and has excellent long-term storage stability. It is effective for the treatment.

In the present invention, the microorganism-derived antigen protein gene includes ESAT-6 gene, Ag85A gene, Ag85B gene, Ag85C gene, CFP-10 gene, TB7.7 gene, MPT51 gene, HSP65 gene and other tuberculosis bacteria, Pseudomonas aeruginosa, P24 antigen gene of rabies virus G protein gene, prM protein and E protein gene of West Nile virus, those derived from bacteria such as pneumococci and streptococci, adenovirus capsid antigens hexon, penton, and fiber genes , Viruses derived from viruses such as the M1 protein and NP protein gene of influenza virus, and those derived from microorganisms such as rickettsia, chlamydia, protozoa, etc., which are partially deleted or partially replaced The tongue, etc. It refers to a gene that encodes a protein or peptide exhibiting an antigenic function of the click quality. For example, ESAT-6 gene means a gene encoding Mycobacterium tuberculosis 6 kDa early secreted antigenic target, and Ag85B gene means a secreted protein of Mycobacterium tuberculosis having 30 kDa mycolyl transferase activity, antigen 85B. In the present invention, immunostimulatory cytokine genes such as GM-CSF, IL-2, and IL-12 may be used at the same time as the microorganism-derived antigen protein gene. Cytokine genes such as GM-CSF, IL-2, and IL-12, in addition to genes encoding GM-CSF, IL-2, and IL-12, those that have been partially deleted or replaced Means a gene encoding a protein or peptide that exhibits the function of GM-CSF, IL-2 or IL-12, and animals such as human GM-CSF, IL-2, IL-12 and dogs and cats GM-CSF, IL-2, and IL-12 are also meant.
When both these microorganism-derived antigen protein gene and immunostimulatory cytokine gene are used, for example, a microorganism-derived antigen protein gene expression cassette and an immunostimulatory cytokine gene expression cassette inserted into a vector in tandem can be used. . Further, a complex containing both a plasmid encoding a microorganism-derived antigen protein gene and a plasmid encoding an immunostimulatory cytokine gene, or a mixture of these complexes may be used.
In the present invention, the vector means a nucleic acid molecule that is used in gene recombination techniques to amplify, maintain, and introduce recombinant DNA, and is generally used as a plasmid vector such as pCMV, pcDNA, pACT; A cosmid vector; means a vector usually used in the art, such as an artificial chromosome vector such as a PAC vector, a YAC vector, and a BAC vector.
Furthermore, in the present invention, the gene introduction reagent means a reagent used for the purpose of introducing a gene, for example, a cationic polymer, a cationic lipid, an anionic polymer, etc., which are usually used. It also means a combination of.
A vector encoding a gene in the complex of the present invention (hereinafter referred to as “nucleic acid etc.”) forms a complex by ionic bonding with a cationic polymer or a cationic lipid or an aggregate containing the same. When an anionic polymer is added, the anionic polymer is ionically bonded to the cationic polymer or the cationic lipid. Depending on the mixing ratio, mixing order, etc., these can form a complex mainly covered with an anionic polymer.
The cationic polymer that can be used in the complex of the present invention is a naturally-derived or synthetic polymer having a positively charged molecular weight of about 1,000 to 3,000,000, and a functional group that can form a complex with DNA in water. A polymer having a plurality of, preferably 5 or more in a molecule can be used. Examples of such functional groups include amino groups or ammonium groups which may be substituted, or salts thereof (these groups are And an organic amino group such as an imino group, an imidazolyl group, and a guanidino group, which may be mono- or polysubstituted by an alkyl group having 1 to 6 carbon atoms, a phenyl group, or the like. Examples of such cationic polymers include positively charged proteins and polypeptides; positively charged dendrimers; positively charged synthetic polymers; and positively charged polysaccharide derivatives, or grafts thereof. Or a block copolymer and a salt thereof, and a combination thereof.
The molecular weight of the positively charged protein or positively charged polypeptide that can be used as the cationic polymer in the complex of the present invention is preferably about 1,000 to 500,000. Specific examples of such proteins and polypeptides include proteins and polypeptides such as protamine, histone, HelΔ1, gelatin, and polyamino acids containing positively charged amino acid residues are also exemplified. It can be illustrated. Specific examples of such a polyamino acid containing a positively charged amino acid residue include poly-L-lysine, polyarginine, polyornithine and the like. Examples of salts of these proteins and polypeptides include hydrochlorides, sulfates, phosphates, borates and the like.
The positively charged dendrimer having a functional group as described above which can be used as a cationic polymer is an amino group or ammonium group which may be substituted at the terminal or inside of a branched molecular chain or a salt thereof ( These groups are dendrimers having, for example, a mono- or poly-substituted group having, for example, an alkyl group having 1 to 6 carbon atoms or a phenyl group, and the molecular weight thereof is preferably about 1,000 to 500,000. Specific examples of dendrimers include polyamidoamine dendrimers and polylysine dendrimers. Examples of the dendrimer salt include hydrochloride, sulfate, phosphate, borate and the like.
A positively charged synthetic polymer that can be used as a cationic polymer is a synthetic polymer having a plurality of, preferably five or more, functional groups that can form a complex with DNA in water as described above. The synthetic polymer preferably has a molecular weight of 1,000 to 3,000,000. Specific examples of the synthetic polymer include polyethyleneimine (including linear polyethyleneimine or polybranched ethyleneimine), a polymer or copolymer of 2-dimethylaminoethyl methacrylate, and a polymer of 2-trimethylaminoethyl methacrylate. Examples thereof include a polymer or a copolymer, polyvinyl imidazole, a polymer having an ethylenediamine structure in the side chain and a copolymer thereof, or a salt thereof. The molecular weight of polyethyleneimine, which is an example of a synthetic polymer, is preferably about 1,000 to 500,000, more preferably about 5,000 to 200,000, and most preferably about 10,000 to 100,000. Or it is a block copolymer obtained by combining shorter polyethyleneimine. Examples of the polyethyleneimine salt include hydrochloride, sulfate, phosphate, borate and the like.
The positively charged polysaccharide derivative that can be used as the cationic polymer has a plurality of, preferably 5 or more, functional groups capable of forming a complex with DNA in water, and has a molecular weight of preferably 1000 to The polysaccharide derivative is 3 million, more preferably 5000 to 500,000. Specific examples of such polysaccharides include chitosan and dextran derivatives introduced with the above functional groups. Of these, the molecular weight of chitosan is preferably about 1,000 to 500,000, more preferably about 5,000 to 200,000, and most preferably about 10,000 to 100,000. Examples of the salt of chitosan include hydrochloride and acetate. The molecular weight of the dextran derivative is preferably 3,000 to 1,000,000. Specific examples of such dextran derivatives include diethylaminoethyl-dextran.
The cationic polymer may be a polymer that is positively charged by introducing a functional group such as an amino group into a conventionally non-positively charged polymer. Moreover, even if it is not normally positively charged, it can be used as long as it is positively charged at the time of complex formation, and if necessary, it can be further modified with a sugar chain, oligopeptide, antibody or the like. It may be.
Cationic lipids (including cationic cholesterol derivatives) that can be used in the complex of the present invention include DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol), DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DMRI (N- (1,2-dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- ( 2- (sperminecarboxamido) ethyl) -N, N-dimethylammonium trifluoro Cetate), DOTAP (N- (1- (2,3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride), or DOTMA (N- (1- (2,3-dioleoyloxy) ) Propyl) -N, N, N-trimethylammonium chloride), and their salts with different acids, and combinations thereof.
Moreover, as an aggregate | assembly containing a cationic lipid, what mixed the said cationic lipid (for example, DOSPA), and neutral substances, such as DOPE (dioleoylphosphatidylethanolamine), cholesterol, etc. can be used. For example, as an aggregate containing a cationic lipid, lipofectamine (3: 1 w / w mixture liposome of DOSPA and DOPE), lipofectin (1: 1 w / w mixture liposome of DOTMA and DOPE), or a mixture thereof, etc. Can be preferably mentioned. Furthermore, antibodies against podoplanin, podocalyxin, kitalan sulfate, ganglioside, podoplanin-CLEC-2 receptor (C-type lectin-like receptor-2), MUC, or various glycoproteins specific to tumor cells Or a cationic polymer to which an anti-HER-2 antibody such as rituximab (anti-CD20 antibody) or trastuzumab (Herceptin), an anti-EGFR antibody, omnitag, or the like, which is an antibody drug, is used to impart targeting properties. it can.
In the composite of the present invention, the cationic polymer includes polyethyleneimine; protamine; HelΔ1; dendrimers such as polyamidoamine dendrimer and polylysine dendrimer; chitosan; polymer or copolymer of 2-dimethylaminoethyl methacrylate; 2-trimethyl A polymer or copolymer of aminoethyl methacrylate can be preferably used, and polyethyleneimine, polyamidoamine dendrimer, polylysine dendrimer, and chitosan can be particularly preferably used. Moreover, as a cationic lipid or an aggregate containing the same, lipofectamine (the above-mentioned 3: 1 w / w mixture liposome of DOSPA and DOPE) can be preferably used.
The anionic polymer used in the composite of the present invention is a negatively charged, naturally-occurring or synthetic polymer having a molecular weight of about 5 to 4 million, containing an anionic group in the molecule. A polymer having a plurality of, preferably 5 or more, functional groups capable of forming a complex with a cation can be used. Examples of such functional groups include a carboxyl group, -OSO, and the like. ₃ H group, -SO ₃ H groups, phosphate groups, and salts thereof can be mentioned. Such anionic polymers include amphoteric polymers.
In the composite of the present invention, the anionic polymer includes a carboxyl group, -OSO. ₃ H group, -SO ₃ Polysaccharides or derivatives thereof having functional groups selected from H groups, phosphate groups, and salts thereof; polyamino acids containing amino acid residues having negatively charged side chains; PEG derivatives having carboxyl side chains; carboxyl Group, -OSO ₃ H group, -SO ₃ Synthetic polymer having a functional group selected from H group, phosphoric acid group, and salts thereof; carboxyl group, -OSO ₃ H group, -SO ₃ H group, phosphate group, and functional group selected from salts thereof, and optionally substituted amino group or ammonium group or a salt thereof (for example, these groups are alkyl groups having 1 to 6 carbon atoms, phenyl Macromolecules which may be mono- or polysubstituted), as well as combinations thereof, can be used.
As the polysaccharide having a functional group as described above that can be used as an anionic polymer in the complex of the present invention or a derivative thereof, glycosaminoglycan can be preferably used. The molecular weight of such glycosaminoglycan is preferably 1,000 to 4,000,000, more preferably 4,000 to 3,000,000. Specific examples of such glycosaminoglycans include hyaluronic acid, chondroitin, chondroitin sulfate, keratan sulfate, heparin, dermatan sulfate and the like. Of these, chondroitin sulfate and hyaluronic acid can be preferably used. Chondroitin sulfate can also be used as a salt thereof or a negatively charged derivative. The molecular weight may be 1,000 or more, preferably 3,000 or more, and more preferably 7,000 to 50,000. Examples of the salt of chondroitin sulfate include sodium salt, potassium salt, ammonium salt and the like. Examples of chondroitin sulfate derivatives include those obtained by introducing polyethylene glycol, peptides, sugars, proteins, iodic acid, antibodies or parts thereof into chondroitin sulfate, and introduce spermine, spermidine, etc. And zwitterionic derivatives having a positively charged moiety. Hyaluronic acid can also be used as its salt or a negatively charged derivative. The molecular weight may be 5,000 or more, preferably 10,000 or more, and more preferably 100,000 to 3,000,000. Examples of the salt of hyaluronic acid include sodium salt, potassium salt, ammonium salt and the like. In addition, examples of the derivatives of hyaluronic acid include those obtained by introducing polyethylene glycol, peptides, sugars, proteins, iodic acid, antibodies or parts thereof into hyaluronic acid, and spermine, spermidine, etc. are introduced. And zwitterionic derivatives having a positively charged moiety.
The polyamino acid containing an amino acid residue having a negatively charged side chain that can be used as an anionic polymer in the complex of the present invention is a carboxyl group, -O-SO. ₃ H group, -SO ₃ A polyamino acid having an amino acid residue having a side chain of a group such as an H group, a phosphate group, and a salt thereof, preferably having a molecular weight of 500 to 1,000,000. Specific examples of such polyamino acids include polyglutamic acid and polyaspartic acid.
The PEG derivative having a carboxyl side chain that can be used as an anionic polymer in the complex of the present invention has a plurality of, preferably 5 or more, carboxyl side chains per PEG molecule, 500 or more, preferably 2000 or more, more preferably Is a PEG derivative having a molecular weight of 4000-40000. A PEG derivative having a carboxyl side chain can also be used as a salt thereof or a negatively charged derivative. Examples of these salts include sodium salts, potassium salts, ammonium salts and the like. Specific examples of such PEG derivatives include those described in J. Org. Biometer. Sci. Polymer Edn. Vol. 14, pp 515-531 (2003) and the like.
A carboxyl group that can be used as an anionic polymer in the complex of the present invention, -OSO ₃ H group, -SO ₃ The synthetic polymer having a functional group selected from H group, phosphoric acid group, and salts thereof includes a plurality of, preferably 5 or more, carboxyl groups, —O—SO per molecule. ₃ H group, -SO ₃ A polymer or copolymer having a functional group selected from an H group, a phosphate group, and a salt thereof, preferably a polymer or copolymer having a molecular weight of 5 to 4 million. Specific examples of such a polymer or copolymer include a polymer or copolymer of acrylic acid or methacrylic acid having a molecular weight of 1,000,000 to 3,000,000, a sulfate ester of polyvinyl alcohol, or succinimidylated poly-L-lysine. Etc. can be illustrated.
A carboxyl group that can be used as an anionic polymer in the complex of the present invention, -OSO ₃ H group, -SO ₃ H group, phosphate group, and functional group selected from salts thereof, and optionally substituted amino group or ammonium group or a salt thereof (for example, these groups are alkyl groups having 1 to 6 carbon atoms, phenyl And a polymer having a mono- or poly-substituted group such as a carboxyl group, -OSO per molecule ₃ H group, -SO ₃ A plurality of functional groups selected from H groups, phosphoric acid groups, and salts thereof, preferably 5 or more, and 500 or more having an amino group or ammonium group or a salt thereof optionally substituted as described above The polymer is preferably a polymer having a molecular weight of 2000 or more, more preferably 4000 to 40000. As such a polymer, preferably, a PEG derivative having a carboxyl side chain and the amino group or ammonium group or a salt thereof equal to or less than the carboxyl side chain can be mentioned, specifically, Macromol. Biosci. Vol. 2, pp 251-256 (2002), and the like.
The anionic polymer that can be used in the composite of the present invention may be one that has been negatively charged by introducing a functional group such as a carboxyl group into a conventionally non-negatively charged one. Even if it is not negatively charged normally, it can be used as long as it is negatively charged at the time of complex formation, and if necessary, it may be further modified with sugar chain, oligopeptide, antibody, etc. Good.
In the complex of the present invention, as the anionic polymer, hyaluronic acid, chondroitin sulfate, PEG derivatives having a carboxyl side chain, anionic polymers such as polyacrylic acid or salts thereof can be preferably used. A PEG derivative having a carboxyl side chain or a salt thereof can be particularly preferably used.
Moreover, it is possible to introduce a gene specifically to a target cell by using an anionic polymer having a specific adhesion ability to a target cell for gene introduction. For example, when chondroitin sulfate is used as the anionic polymer, cells having cell surface molecules such as CD44 variants that specifically bind to chondroitin sulfate can be targeted. When hyaluronic acid is used as the anionic polymer, cells having cell surface molecules such as CD44 that specifically bind to hyaluronic acid can be targeted. In addition, many types of tumor cells can be targeted by using an anionic polymer into which an RGD peptide has been introduced, and hepatocytes or liver-derived cells by using an anionic polymer into which a galactose side chain has been introduced. Can be targeted. Furthermore, antibodies against podoplanin, podocalyxin, kitalan sulfate, ganglioside, podoplanin-CLEC-2 receptor (C-type lectin-like receptor-2), MUC, or various glycoproteins specific to tumor cells Or an anionic polymer to which an anti-HER-2 antibody such as rituximab (anti-CD20 antibody) or trastuzumab (Herceptin), an anti-EGFR antibody, omnitag, or the like, which is an antibody drug, is used to impart targeting properties. it can.
In the complex of the present invention, the combination of the cationic polymer or the cationic lipid or the assembly containing the cationic polymer and the anionic polymer includes polyethyleneimine and hyaluronic acid; polyethyleneimine and chondroitin sulfate; polyethyleneimine and carboxyl side chain. PEG derivatives; aggregates containing DOSPA (eg, lipofectamine (3: 1 w / w mixture liposome of DOSPA and DOPE)) and hyaluronic acid; aggregates containing DOSPA (eg, lipofectamine) and PEG derivatives having carboxyl side chains are preferred. be able to.
The molar ratio (negative charge: positive charge ratio) of each charged group of the nucleic acid or the like used in the complex of the present invention and the cationic polymer or cationic lipid or the assembly containing the same is the target cell, nucleic acid, etc. Although it varies depending on the type of polymer or the like, it is preferably 1: 0.8 to 1: 100, preferably 1: 1 to 1:50, more preferably 1: 1.2 to 1:30. The compounding ratio of the nucleic acid and the like here and the cationic polymer or the cationic lipid or the assembly containing the same is a molar ratio of each charged group, specifically, phosphoric acid of nucleic acid, oligonucleic acid, or a derivative thereof. Negative charge by anion: Refers to the molar ratio of the positively charged or functional group capable of being positively charged of a cationic polymer or cationic lipid or an assembly containing the same.
The molar ratio (negative charge: negative charge ratio) between the nucleic acid used in the complex of the present invention and each charged group of the anionic polymer varies depending on the type of the target cell, nucleic acid, etc., anionic polymer, but 1: 0 2 to 1: 1000, preferably 1: 0.2 to 1: 100, and more preferably 1: 1 to 1:60. The compounding ratio of the nucleic acid and the anionic polymer here is a molar ratio of each charged group. Specifically, the negative charge by the phosphate anion of the nucleic acid, oligonucleic acid, or derivative thereof: negative of the anionic polymer It refers to the molar ratio of functional groups that can be charged or negatively charged.
For example, when chondroitin sulfate or hyaluronic acid is used as the anionic polymer, the mixing ratio of nucleic acid or the like to chondroitin sulfate or hyaluronic acid may be 1: 0.2 to 1: 1000, preferably 1: 0.2. ˜1: 100, more preferably 1: 1 to 1:60.
In particular, when polyethyleneimine is used as the cationic polymer and chondroitin sulfate or hyaluronic acid is used as the anionic polymer, the mixing ratio of nucleic acid and the like: polyethyleneimine: chondroitin sulfate (or hyaluronic acid) is 1: 2 to 60: 1 to 240, The ratio is preferably 1: 4 to 24: 1 to 160, more preferably 1: 5 to 20: 2 to 60, and particularly preferably 1: 6 to 14: 2 to 32.
In particular, when lipofectamine (3: 1 w / w mixture liposome of DOSPA and DOPE) is used as an assembly containing a cationic lipid, and chondroitin sulfate or hyaluronic acid is used as an anionic polymer, nucleic acid or the like: lipofectamine: chondroitin sulfate (or hyaluron) Acid) The mixing ratio is 1: 1 to 50: 0.2 to 240, preferably 1: 1.2 to 48: 0.2 to 160, more preferably 1: 1.5 to 30: 0.5. ~ 60, particularly preferably 1: 1.8 to 16: 1 to 32.
The preferred mixing ratio of the nucleic acid and the like contained in the complex of the present invention; the cationic polymer or the cationic lipid or the aggregate containing the same; and the anionic polymer is as described above, but the number of cells into which the nucleic acid or the like is introduced Since the optimum conditions vary depending on the type, the mixing ratio can be appropriately determined by those skilled in the art according to the type of cells, nucleic acids, and the like used.
The complex of the present invention is formed by mixing the above-described nucleic acid or the like; a cationic polymer or a cationic lipid or an aggregate containing the same; and if necessary, an anionic polymer in the above-described mixing ratio. Can be prepared by the step of allowing, if necessary, followed by lyophilization. The order of mixing is [1] nucleic acid or the like; [2] cationic polymer or cationic lipid or aggregate containing the same, [3] order of anionic polymer, or [1] nucleic acid or the like; [2] anion The order of the functional polymer, [3] cationic polymer or cationic lipid or an assembly containing the same is preferred. A nucleic acid or the like is a complex in which a cationic polymer or a cationic lipid or an assembly containing the same is bonded by an ionic bond, and the cationic polymer or the cationic lipid or an assembly including the ion is also ionically bonded to an anionic polymer. It is formed. Alternatively, depending on the blending composition of each component, an anionic polymer mainly coats the outer shell of such a complex structure, and an embodiment having a negative surface potential is formed.
The resulting complex is then lyophilized if necessary. Freeze-drying can be performed under normal freeze-drying conditions. For example, under reduced pressure (preferably 5 to 100 mmHg, more preferably 10 mmHg), outside air temperature -78 ° C to 60 ° C, preferably -30 ° C to 40 ° C. Can be done by drying with. The time required for drying varies depending on the degree of vacuum and the amount of solvent, and is usually completed in 1 hour to 2 days.
The complex of the present invention prepared as described above can be used for various gene therapy and immunotherapy for humans and animals, or creation of experimental animals and cells into which a microorganism-derived antigen protein gene or immunostimulatory cytokine gene is introduced. Can do. When freeze-dried, the complex of the present invention is used as a rehydrate by suspending or dissolving the complex of the present invention in a solvent such as water, physiological saline, buffer solution, glucose solution, medium solution or the like before use. be able to. Upon rehydration, the lyophilized product is suspended or diluted with a solvent, for example, 100 to 10,000 times (by weight) the nucleic acid or derivative thereof. Since different amounts of solvents and different types of solvents can be used before lyophilization, comparatively high concentration suspensions and solutions (for example, solutions containing 1 mg of DNA in 1 ml) can be easily prepared. Can do.
When the nucleic acid or the like is rehydrated in this way, the complex of the present invention specifically includes, for example, a complex of the present invention in which target cells taken out of the body in a well are hydrated. Direct gene such as ex vivo method (vaccine therapy) in which the gene is introduced by treatment with the body and then the cells are returned to the living body to express the target gene, or in vivo, in situ method, etc. Any method commonly used for introducing a nucleic acid or a derivative thereof into a living cell, such as an introduction method, can be used.
In addition, the complex of the present invention can be brought into contact with a cell to which nucleic acid or the like is introduced without being rehydrated after lyophilization, or transferred to an animal to which nucleic acid or the like is introduced, or introduced into nucleic acid or the like. It can also be administered by means such as transplanting within, on or near the target tissue.
The amount of the complex of the present invention applied to the cells varies depending on the introduction method, the type of disease, etc. described above. For example, the amount of nucleic acid or a derivative thereof is 1 to 2 cm in diameter in the ex vivo method or the in situ method. 0.2 to 10 μg / 104 to 7 cells / well per cell, and in vivo method varies greatly depending on the administration site, but for local administration into a tumor, for example, 5 to 1000 μg / cm ³ For administration to organs such as tumors and bladder, for example, 0.1 μg to 100 mg / organ, and for systemic administration, for example, 0.1 ng to 10 mg / Kg body weight.
As an in vivo method for direct administration to a living body, the complex of the present invention or a hydrated lyophilized hydrate of the present invention is injected into a vein, subcutaneous or muscle, abdominal cavity, intratumoral, in the vicinity of a tumor, or the like. Inhaled from the nasal cavity, oral cavity, lungs, etc .; injected directly into the bladder or rectum; administered directly into the affected tissue or nearby blood vessels; or carried on a porous material such as a gel or sponge or a nonwoven fabric Any method of gene therapy technique can be used, such as indwelling.
In addition, even when the lyophilized product hydrate of the present invention is used without rehydration, the above amount of the lyophilized product is obtained by the ex vivo method, the in situ method, or the in vivo method as described above. Can be used.
In the complex of the present invention, the anionic polymer neutralizes the positive charge of the complex of a normal nucleic acid or the like and a cationic polymer or a cationic lipid or an assembly containing the same, and the neutralization thereof. Since the action is retained even after administration to a living body or a cell, interaction such as aggregation between the complex and serum protein, blood cell, extracellular matrix, etc. is blocked. In addition, since enzymatic degradation of derivatives thereof or the like is prevented, nucleic acids are efficiently taken up by cells and their expression efficiency is high.
From the above, the complex of the present invention can be used as a preparation or reagent for introducing a nucleic acid or a derivative thereof, or as a kit for introducing a nucleic acid or a derivative thereof.
The immunostimulatory cytokine gene of the plasmid or derivative thereof used in the present invention can be freely selected from human cytokines, cat cytokines, canine cytokines and the like depending on the animal species of interest.
The promoter of the plasmid or derivative thereof used in the present invention may be a cytomegavirus-derived promoter, a RSV (Rous Sarcoma virus) -derived promoter, or SV40 (simian virus 40 (simian virus 40), depending on the animal species or cell type. )) Origin promoter, Cancer selective promoter such as Evolution Factor 1a promoter, etc.

Curative effect on subcutaneously transplanted B16 cells by intratumoral injection of a plasmid / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex encoding the gene for ESAT-6 (vertical axis: tumor volume mm ³ , horizontal axis: drug administration) Indicates the number of days since the start (the same applies to FIGS. 2 to 6) Curative effect on subcutaneously transplanted B16 cells by intratumoral injection of the plasmid complex encoding the gene of ESAT-6 and the plasmid complex encoding the gene of mouse GM-CSF Curative effect on subcutaneously transplanted B16 cells by intratumoral injection of a plasmid complex encoding the gene of ESAT-6 and a plasmid complex encoding the gene of mouse IL-2 Curative effect on subcutaneously transplanted B16 cells by intratumoral injection of a plasmid complex encoding the gene of ESAT-6 and a plasmid complex encoding the gene of mouse IL-12 Curing effect on subcutaneously transplanted B16 cells by intratumoral injection of plasmid complex encoding Ag85B gene Curative effect on subcutaneously transplanted B16 cells by intratumoral injection of a combination of plasmid complex encoding ESAT-6 gene and plasmid complex encoding Ag85B gene

The present invention will be described more specifically with reference to examples. In addition, these Examples are for demonstrating this invention, Comprising: This invention is not limited at all.
Example 1
Curative effect on subcutaneously transplanted B16 cells by intratumoral injection of plasmid (pDNA-ESAT-6) / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex encoding ESAT-6 gene pDNA-ESAT-6, PEI The three-component complex consisting of CS was administered intratumorally to mice transplanted subcutaneously with B16 derived from mouse melanoma cells, and the tumor growth inhibitory effect was confirmed.
[0065]
As the CS, a shark-derived product manufactured by Seikagaku Corporation was used. The PBS used was a solution of Phosphate Buffered Salts (Tablet) manufactured by Roman Industries in distilled ion-exchanged water. As the PEI, a linear PEI manufactured by Polyscience, Polyethyleneimine “Max” (Mw = 40000) was used. The same applies to the following embodiments.
[Operating procedure]
[1] The synthesis of pDNA-ESAT-6 was requested from Takara Bio Inc. As an expression vector, pcDNA3.1 (+) of Invitrogen Corporation was used.
[2] B16 cells were seeded in a cell culture bottle and incubated using MEM medium containing 10% FBS, 25 U penicillin and 25 μg streptomycin to prepare cells for transplantation.
[3] The culture medium cultured immediately before transplantation was removed, a trypsin solution was added, and the cells were detached.
[4] 20 × 10 ⁵ B16 melanoma cells were transplanted subcutaneously into the abdomen of 5-week-old male C57BL / 6J mice.
[5] Several days before the introduction, a plasmid complex was prepared by the following method.
119 μl of an aqueous solution containing 594 μg of CS is added to 4820 μl of 7.4 mM phosphate buffer (pH 7.4) containing pDNA-ESAT-6 (100 μg), and then 59 μl of an aqueous solution containing 293.5 μg of PEI is added and stirred. And left at room temperature for 20 minutes. Thereafter, 50 μl of 10% dextran solution was added, and the mixture was further left for 10 minutes and then frozen at −80 ° C. or −30 ° C. Then, it lyophilized | freeze-dried and the composite_body | complex of this invention was prepared.
[6] When the tumor size of the tumor-bearing mouse model prepared in [4] becomes 3 mm or more in the major axis, 250 μl of pure water is added to the freeze-dried product prepared in [5], and the resultant is sufficiently dissolved and dispersed. And administered into the tumor tissue.
[7] The tumor size was measured once a day.
[result]
The results are shown in FIG. Here, the tumor size in the figure is calculated as (4/3) x minor axis x minor axis x major axis x (1/8) x 3.14.
In the control group to which nothing was administered, there was a sudden increase in tumor, whereas in the mice to which the pDNA-ESAT-6 / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex was administered locally in the tumor, Clear suppression of tumor growth was observed.
Example 2
The curative effect with respect to the subcutaneous transplantation B16 cell by the local injection of the tumor which used the plasmid (pDNA-GM-CSF) composite_body | complex which coded the gene of mouse | mouth GM-CSF in the pDNA-ESAT-6 composite_body | complex.
A pDNA-GM-CSF / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex was prepared in the same manner as in Example 1, and the pDNA-ESAT-6 / polyethyleneimine (PEI) / chondroitin sulfate (prepared in Example 1) CS) complex and lyophilized. After rehydration, B16 derived from mouse melanoma cells was subcutaneously administered to mice transplanted subcutaneously, and the tumor growth inhibitory effect was confirmed.
[Operating procedure]
[1] A tumor-bearing model mouse, pDNA-ESAT-6 / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex, and its pDNA-GM-CSF / polyethyleneimine (PEI) / chondroitin sulfate as in Example 1. A freeze-dried product of a mixture with the (CS) complex was prepared.
[2] When the tumor size of the tumor-bearing mouse model becomes 3 mm or more in the major axis, 250 μl of pure water is added to the above-mentioned two types of freeze-dried products, and the mixture is sufficiently dissolved and dispersed, and administered into the tumor tissue. did. The mixing ratio was pDNA-ESAT-6 complex: pDNA-GM-CSF complex = 4: 1 (weight ratio). One dose was such that the total amount of plasmid was 100 μg.
[3] Tumor size was measured once a day.
[result]
The results are shown in FIG. Here, the tumor size in the figure is calculated as (4/3) x minor axis x minor axis x major axis x (1/8) x 3.14.
Compared to the group to which only the pDNA-ESAT-6 complex or only the pDNA-GM-CSF complex was administered, the mice in which both were mixed and administered into the tumor tissue had a greater tumor growth inhibitory effect and a synergistic effect Was recognized.
Example 3
The curative effect on subcutaneously transplanted B16 cells by intratumoral injection of a plasmid (pDNA-IL-2) complex that encodes the mouse mouse IL-2 gene in combination with the pDNA-ESAT-6 complex.
A pDNA-IL-2 / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex was prepared in the same manner as in Example 1, and the pDNA-ESAT-6 / polyethyleneimine (PEI) / chondroitin sulfate (prepared in Example 1) CS) complex and lyophilized. After rehydration, B16 derived from mouse melanoma cells was subcutaneously administered to mice transplanted subcutaneously, and the tumor growth inhibitory effect was confirmed.
[Operating procedure]
[1] Tumor-bearing model mouse, pDNA-ESAT-6 / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex, and pDNA-IL-2 / polyethyleneimine (PEI) / chondroitin sulfate as in Example 1 A freeze-dried product of a mixture with the (CS) complex was prepared.
[2] When the tumor size of the tumor-bearing mouse model becomes 3 mm or more in the major axis, 250 μl of pure water is added to the above-mentioned two types of freeze-dried products, and the mixture is sufficiently dissolved and dispersed, and administered into the tumor tissue. did. The mixing ratio was pDNA-ESAT-6 complex: pDNA-IL-2 complex = 2: 3 (weight ratio). One dose was such that the total amount of plasmid was 100 μg.
[3] Tumor size was measured once a day.
[result]
The results are shown in FIG. Here, the tumor size in the figure is calculated as (4/3) x minor axis x minor axis x major axis x (1/8) x 3.14.
Compared to the group to which only the pDNA-ESAT-6 complex or only the pDNA-IL-2 complex was administered, the mice in which both were mixed and administered into the tumor tissue had a greater tumor growth inhibitory effect and a synergistic effect Was recognized.
Example 4
The curative effect on subcutaneously transplanted B16 cells by intratumoral injection of a plasmid (pDNA-IL-12) complex encoding a mouse mouse IL-12 gene in combination with a pDNA-ESAT-6 complex.
A pDNA-IL-12 / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex was prepared in the same manner as in Example 1, and the pDNA-ESAT-6 / polyethyleneimine (PEI) / chondroitin sulfate (prepared in Example 1) CS) complex and lyophilized. After rehydration, B16 derived from mouse melanoma cells was subcutaneously administered to mice transplanted subcutaneously, and the tumor growth inhibitory effect was confirmed.
[Operating procedure]
[1] Tumor-bearing model mouse, pDNA-ESAT-6 / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex, and pDNA-IL-12 / polyethyleneimine (PEI) / chondroitin sulfate as in Example 1 A freeze-dried product of a mixture with the (CS) complex was prepared.
[2] When the tumor size of the tumor-bearing mouse model becomes 3 mm or more in the major axis, 250 μl of pure water is added to the above-mentioned two types of freeze-dried products, and the mixture is sufficiently dissolved and dispersed, and administered into the tumor tissue. did. The mixing ratio was pDNA-ESAT-6 complex: pDNA-IL-12 complex = 3: 2 (weight ratio). One dose was such that the total amount of plasmid was 100 μg.
[3] Tumor size was measured once a day.
[result]
The results are shown in FIG. Here, the tumor size in the figure is calculated as (4/3) x minor axis x minor axis x major axis x (1/8) x 3.14.
Compared to the group to which only the pDNA-ESAT-6 complex or only the pDNA-IL-12 complex was administered, the mice administered with both of them mixed and administered into the tumor tissue had a greater tumor growth inhibitory effect and a synergistic effect Was recognized.
Example 5
Healing effect on subcutaneously transplanted B16 cells by intratumoral injection of Ag85B gene-encoding plasmid (pDNA-Ag85B) / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex Three components consisting of pDNA-Ag85B, PEI and CS The above complex was administered intratumorally to mice transplanted subcutaneously with B16 derived from mouse melanoma cells, and the tumor growth inhibitory effect was confirmed.
[Operating procedure]
[1] The synthesis of pDNA-Ag85B was requested from Takara Bio Inc. As an expression vector, pcDNA3.1 (+) of Invitrogen Corporation was used.
[2] In the same manner as in Example 1, a tumor-bearing model mouse, a lyophilized product of pDNA-Ag85B / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex was prepared.
[2] When the tumor size of the tumor-bearing mouse model becomes 3 mm or more in the major axis, 250 μl of pure water is added to the above-mentioned two types of freeze-dried products, and the mixture is sufficiently dissolved and dispersed, and administered into the tumor tissue. did.
[3] Tumor size was measured once a day.
[result]
The results are shown in FIG. Here, the tumor size in the figure is calculated as (4/3) x minor axis x minor axis x major axis x (1/8) x 3.14.
In the control group to which nothing was administered, rapid tumor growth was observed, whereas in the mice to which the pDNA-Ag85B / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex was administered locally, it was clear. Tumor growth suppression was observed.
Example 6
Curing effect on subcutaneously transplanted B16 cells by intratumoral injection of a tumor using a combination of pDNA-Ag85B complex and mouse mouse pDNA-ESAT-6 complex.
A pDNA-Ag85B / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex was prepared in the same manner as in Example 5, and the pDNA-ESAT-6 / polyethyleneimine (PEI) / chondroitin sulfate (CS) prepared in Example 1 was used. Mixed with the complex and lyophilized. After rehydration, B16 derived from mouse melanoma cells was subcutaneously administered to mice transplanted subcutaneously, and the tumor growth inhibitory effect was confirmed.
[Operating procedure]
[1] Tumor-bearing model mouse as in Example 1, pDNA-ESAT-6 / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex, pDNA-Ag85B / polyethyleneimine (PEI) / chondroitin sulfate (CS) complex The body, and the lyophilized body of those mixtures were prepared.
[2] When the tumor size of the tumor-bearing mouse model becomes 3 mm or more in the major axis, 250 μl of pure water is added to the above-mentioned two types of freeze-dried products, and the mixture is sufficiently dissolved and dispersed, and administered into the tumor tissue. did. The mixing ratio was pDNA-ESAT-6 complex: pDNA-Ag85B complex = 1: 1 (weight ratio). One dose was such that the total amount of plasmid was 100 μg.
[3] Tumor size was measured once a day.
[result]
The results are shown in FIG. Here, the tumor size in the figure is calculated as (4/3) x minor axis x minor axis x major axis x (1/8) x 3.14.
Compared to the group administered only with the pDNA-ESAT-6 complex or only the pDNA-Ag85B complex, the mice administered with both mixed and administered in the tumor tissue had a greater tumor growth inhibitory effect and a synergistic effect was observed. It was.

Claims (41)

1. A complex comprising a vector encoding a microorganism-derived antigen protein gene and a gene introduction reagent.
2. The complex according to claim 1, wherein the microorganism is Mycobacterium tuberculosis.
3. The complex according to claim 2, wherein the Mycobacterium tuberculosis-derived antigenic protein is ESAT-6 (Mycobacterium tuberculosis 6 kDa early secreted antigenic target) and / or Ag85B.
4. The complex according to any one of claims 1 to 3, wherein the vector is a plasmid vector.
5. The complex according to any one of claims 1 to 4, wherein the gene introduction reagent comprises one or more compounds selected from a cationic polymer, a cationic lipid, and an anionic polymer.
6. 6. The complex according to claim 5, wherein the gene introduction reagent comprises a cationic polymer or a cationic lipid and an anionic polymer.
7. The cationic polymer is polyethyleneimine (linear polyethyleneimine or polybranched ethyleneimine), 2-dimethylaminoethyl methacrylate polymer or copolymer, 2-trimethylaminoethyl methacrylate polymer or copolymer, Polyamidoamine dendrimer, polylysine dendrimer, protamine, histone, HelΔ1, gelatin, poly-L-lysine, polyarginine, polyornithine, polyvinyl imidazole, polymers having an ethylenediamine structure in the side chain and copolymers thereof, or salts thereof The complex according to claim 5 or 6, wherein one or more are selected from.
8. Cationic lipids include DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol, DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DMRI (N -(1,2-Dimyristyloxyppa-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS Heptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- (2- (sperminecarboxamide) -N, N-dimethylammonium trifluoroacetate), DOTAP (N- (1- (2,3-dioleyloxy) propyl) -N, N, N-to Methylammonium chloride) and DOTMA (N- (1- (2,3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride) and their different salts with different acids Item 7. The complex according to Item 5 or 6.
9. Anionic polymer is a PEG derivative having a carboxyl side chain, a polymer or copolymer of acrylic acid or methacrylic acid, a sulfate ester of polyvinyl alcohol, succinimidylated poly-L-lysine, polyglutamic acid, polyaspartic acid, hyaluronic acid 9. The complex according to any one of claims 5 to 8, which is selected from one or more of chondroitin, chondroitin sulfate, keratan sulfate, heparin, deltaman sulfate or a derivative thereof, and a salt thereof.
10. 10. The composite according to any one of claims 1 to 9, which has been lyophilized.
11. A pharmaceutical comprising a vector encoding a microorganism-derived antigen protein gene and a gene introduction reagent.
12. The medicament according to claim 11, wherein the microorganism is Mycobacterium tuberculosis.
13. 13. The medicine according to claim 12, wherein the Mycobacterium tuberculosis-derived antigenic protein is ESAT-6 (M. tuberculosis 6 kDa early secreted antigenic target) and / or Ag85B.
14. The medicine according to any one of claims 11 to 13, wherein the vector is a plasmid vector.
15. 15. The medicament according to claim 11, wherein the gene introduction reagent comprises one or more compounds selected from a cationic polymer, a cationic lipid, and an anionic polymer.
16. The medicament according to claim 15, wherein the gene introduction reagent comprises a cationic polymer or a cationic lipid and an anionic polymer.
17. The cationic polymer is polyethyleneimine (linear polyethyleneimine or polybranched ethyleneimine), 2-dimethylaminoethyl methacrylate polymer or copolymer, 2-trimethylaminoethyl methacrylate polymer or copolymer, Polyamidoamine dendrimer, polylysine dendrimer, protamine, histone, HelΔ1, gelatin, poly-L-lysine, polyarginine, polyornithine, polyvinyl imidazole, polymers having an ethylenediamine structure in the side chain and copolymers thereof, or salts thereof The medicine according to claim 15 or 16, which is selected from one or more of the following.
18. Cationic lipids include DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol, DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DMRI (N -(1,2-Dimyristyloxyppa-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS Heptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- (2- (sperminecarboxamide) -N, N-dimethylammonium trifluoroacetate), DOTAP (N- (1- (2,3-dioleyloxy) propyl) -N, N, N-to Methylammonium chloride) and DOTMA (N- (1- (2,3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride) and their different salts with different acids Item 15. A medicine according to Item 15 or 16.
19. Anionic polymer is a PEG derivative having a carboxyl side chain, a polymer or copolymer of acrylic acid or methacrylic acid, a sulfate ester of polyvinyl alcohol, succinimidylated poly-L-lysine, polyglutamic acid, polyaspartic acid, hyaluronic acid The medicament according to any one of claims 15 to 18, which is selected from one or more of chondroitin, chondroitin sulfate, keratan sulfate, heparin, deltaman sulfate or a derivative thereof, and a salt thereof.
20. 20. The pharmaceutical according to any one of claims 11 to 19, which has been subjected to a freeze-drying treatment.
21. Pharmaceuticals comprising a vector encoding a microorganism-derived antigen protein gene and a complex containing a gene transfer reagent, and a combination of a vector encoding an immunostimulatory cytokine gene and a complex containing a gene transfer reagent.
22. The pharmaceutical according to claim 21, wherein the microorganism is Mycobacterium tuberculosis.
23. 23. The medicament according to claim 22, wherein the Mycobacterium tuberculosis-derived antigenic protein is ESAT-6 (M. tuberculosis 6 kDa early secreted antigenic target) and / or Ag85B.
24. The medicament according to any one of claims 21 to 23, wherein the immunostimulatory cytokine is one or more cytokines selected from interleukin-2 (IL-2), interleukin-12 (IL-12), and GM-CSF.
25. A method for treating cancer, comprising administering an effective amount of a complex comprising a vector encoding a microorganism-derived antigen protein gene and a gene introduction reagent.
26. The method according to claim 25, wherein the microorganism is Mycobacterium tuberculosis.
27. 27. The method according to claim 26, wherein the Mycobacterium tuberculosis-derived antigenic protein is ESAT-6 (Mycobacterium tuberculosis 6 kDa early secreted antigenic target) and / or Ag85B.
28. 28. The treatment method according to claim 25, wherein the vector is a plasmid vector.
29. 29. The treatment method according to claim 25, wherein the gene introduction reagent comprises one or more compounds selected from a cationic polymer, a cationic lipid, and an anionic polymer.
30. 30. The treatment method according to claim 29, wherein the gene introduction reagent comprises a cationic polymer or a cationic lipid and an anionic polymer.
31. The cationic polymer is polyethyleneimine (linear polyethyleneimine or polybranched ethyleneimine), 2-dimethylaminoethyl methacrylate polymer or copolymer, 2-trimethylaminoethyl methacrylate polymer or copolymer, Polyamidoamine dendrimer, polylysine dendrimer, protamine, histone, HelΔ1, gelatin, poly-L-lysine, polyarginine, polyornithine, polyvinyl imidazole, polymers having an ethylenediamine structure in the side chain and copolymers thereof, or salts thereof 31. The treatment method according to claim 29 or 30, wherein one or more are selected from.
32. Cationic lipids include DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol, DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DMRI (N -(1,2-Dimyristyloxyppa-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS Heptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- (2- (sperminecarboxamide) -N, N-dimethylammonium trifluoroacetate), DOTAP (N- (1- (2,3-dioleyloxy) propyl) -N, N, N-to Methylammonium chloride) and DOTMA (N- (1- (2,3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride) and their different salts with different acids Item 31. The treatment method according to Item 30 or 30.
33. Anionic polymer is a PEG derivative having a carboxyl side chain, a polymer or copolymer of acrylic acid or methacrylic acid, a sulfate ester of polyvinyl alcohol, succinimidylated poly-L-lysine, polyglutamic acid, polyaspartic acid, hyaluronic acid The therapeutic method according to any one of claims 29 to 32, which is selected from one or more of chondroitin, chondroitin sulfate, keratan sulfate, heparin, deltaman sulfate or a derivative thereof, and a salt thereof.
34. The treatment method according to any one of claims 25 to 33, which has been lyophilized.
35. A cancer characterized by administering an effective amount of a combination of a vector encoding a microorganism-derived antigen protein gene and a gene transfer reagent, and a vector encoding an immunostimulatory cytokine gene and a complex containing a gene transfer reagent Treatment methods.
36. 36. The treatment method according to claim 35, wherein the microorganism is Mycobacterium tuberculosis.
37. The method according to claim 36, wherein the Mycobacterium tuberculosis-derived antigenic protein is ESAT-6 (Mycobacterium tuberculosis 6 kDa early secreted antigenic target) and / or Ag85B.
38. The method according to claims 35 to 37, wherein the immunostimulatory cytokine is one or more cytokines selected from interleukin-2 (IL-2), interleukin-12 (IL-12), and GM-CSF.
39. The complex, pharmaceutical, or cancer treatment method according to any one of claims 1 to 7, 9 to 17, 19 to 31, and 33 to 38, wherein the cationic polymer is polyethyleneimine.
40. 40. The complex, medicament or cancer treatment method according to any one of claims 1 to 39, wherein the anionic polymer is chondroitin sulfate.
41. 40. The complex, medicament or cancer treatment method according to any one of claims 1 to 39, wherein the anionic polymer is hyaluronic acid.

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