WO2013085021A1

WO2013085021A1 - Composition for enhancing antibody production

Info

Publication number: WO2013085021A1
Application number: PCT/JP2012/081752
Authority: WO
Inventors: 利夫有安; まどか谷合; 大樹長友; 恒孝太田
Original assignee: 株式会社林原
Priority date: 2011-12-09
Filing date: 2012-12-07
Publication date: 2013-06-13
Also published as: JPWO2013085021A1

Abstract

The present invention addresses the problem of providing a novel mucosal adjuvant that is able to enhance antibody production, including that of antigen-specific secreted immunoglobulin A antibodies, by effectively enhancing mucosal immunity when an antigen is administered to the mucosal tissue, and that is stable and safe even when applied in vivo; and of providing a composition for enhancing antibody production in which the active ingredient is the adjuvant and an antigen. The problem is solved by providing a mucosal adjuvant in which the active ingredient is a tumor necrosis factor-α (TNF-α)-cholesterol pullulan complex, and a composition for enhancing antibody production in which the active ingredient is the adjuvant and an antigen.

Description

Composition for enhancing antibody production

The present invention relates to an adjuvant and a composition for enhancing antibody production containing such an adjuvant and an antigen. More specifically, the present invention relates to a safe and safe composition containing a Thumor necrosis factor-α-cholesterol pullulan complex as an active ingredient. The present invention relates to an adjuvant for mucosal administration for efficient enhancement of immune activity and a composition for mucosal administration for enhancement of antibody production containing such an adjuvant and an antigen.

In recent years, with the elucidation of the immune mechanism on the mucosal surface, development of highly effective mucosal immune vaccines has been promoted. Vaccines are most effective in preventing infection with pathogenic microorganisms. However, conventionally used vaccines for subcutaneous inoculation mainly use serum immunoglobulin G antibody (hereinafter, immunoglobulin is abbreviated as “Ig”). In some cases, the prevention of the onset and the prevention of the seriousness are more important than the prevention of infection. In contrast, in the case of mucosal immune vaccines, antigens are secreted in systemic mucosal tissues such as the nasal cavity and respiratory tract by administration through the mucosal tissue route such as the oral cavity and nasal cavity (hereinafter referred to as “mucosal administration”) Type immunoglobulin A antibody (hereinafter sometimes simply referred to as “secreted IgA antibody”) is produced. In addition, IgA antibodies are said to have higher cross-protective ability than IgG antibodies, and the main infection site of pathogenic microorganisms is the mucosa. Therefore, mucosal immune vaccines that excel in secretory IgA antibody production are also effective in protecting infections. Exhibits excellent effects. For example, the outer membrane proteins of Haemophilus influenzae (Non-patent Document 1), which is a causative bacterium such as respiratory tract infections, and Streptococcus mutans, which is a causative bacterium of dental caries ( Non-Patent Document 2) is known to induce immune responses in both mucosal and systemic systems by nasal administration, and these proteins are immune responses against pathogenic microorganisms in mucosal systems. Since it can be induced, clinical application is expected as a new vaccine for protection against infection. In addition, antigens used as vaccines are shifting from conventional attenuated live vaccines and whole cell vaccines to component vaccines and component vaccines in terms of safety.

However, mucosal vaccines, particularly component vaccines and component vaccines, are weakly immunogenic and require administration of a larger amount of antigen compared to subcutaneous vaccines. In addition, administration of a large amount of antigen to mucosal tissue may cause tolerance (unresponsiveness) of the immune response. From these points, in order to induce an effective immune response by mucosal administration, it is indispensable to use an adjuvant in combination with a safe and effective immune response.

Various mucosal administration adjuvants including cholera toxin and microbial cell components have been proposed (see, for example, Patent Document 1). In addition, utilization of heat-resistant toxins of Escherichia coli with reduced toxicity and B subunit of verotoxin 1 has been proposed (see, for example, Patent Document 2).

However, although these known mucosal administration adjuvants are excellent in terms of immunity enhancement, there are problems such as the toxicity and the causal relationship with side effects such as nerve paralysis have not been completely denied. In addition, bacterial cell components that are metabolized and decomposed in vivo release a wide variety of physiologically active substances typified by cytokines, while conversely, to induce an immune response in a specific direction, Poor specificity of releasing only active substance.

In order to eliminate such drawbacks, a method of using cytokines such as interferon or Tsumore necrosis factor-alpha (hereinafter abbreviated as “TNF-α”) as an adjuvant has also been proposed. (For example, refer to Patent Document 3 and Patent Document 4). However, since cytokines are proteins / peptides, they are rapidly deactivated or disappeared in vivo in the order of minutes to hours, and lack a sustained action. In order to solve this problem, a method of frequently administering cytokines to a living body, and a method of administering antigens and cytokines in a DDS format using a colloidal dispersion system such as a polymer or liposome have been proposed, Specifically disclosed is a systematic immune enhancement method in which cytokines such as granulocyte / monocyte-colony stimulating factor (GM-CSF) and interferon-γ are encapsulated in microspheres prepared by crosslinking gelatin and used as DDS. However, there are cases where these methods and preparations have problems in safety and stability.

In addition, TNF-α is not susceptible to degradation when administered to a living body by replacing the lysine residue in the molecule with another amino acid, and has a mucosal administration adjuvant activity similar to that of cholera toxin B subunit. It is known (Non-Patent Document 4). However, since such a mutant TNF-α has higher antigenicity to humans than the wild type, when administered to humans as an adjuvant, an antibody against the mutant TNF-α is produced, and as a result, an adjuvant with the mutant TNF-α. Risks that the effect is reduced or disappeared, and that when mutant TNF-α is administered again as an adjuvant, side effects such as inflammation and allergic symptoms are more likely to be induced than when wild type is used. There is.

Patent Document 1: Japanese Translation of PCT International Publication No. 2008-546658 Patent Document 2: JP 2003-321392 A Patent Document 3: JP 2010-502679 A Patent Document 4: US Patent No. 5,861,159 5: International Publication WO98 / 09650 Pamphlet Patent Document 6: International Publication WO2006 / 077724 Pamphlet

Non-Patent Document 1: Kurono, Y. et al. et al. , “J. Immunol. 161, 4115-4121 (1998)
Non-Patent Document 2: Saito, M .; et al. , “J. Infect. Dis. 183, 823-826 (2001)
Non-Patent Document 3: Adlelet al. , “Cancer Biother. 10: 293-306 (1995)
Non-Patent Document 4: Hiroyuki Amuro et al., “YAKUGAKU ZASSHI”, Vol. 130, No. 1, 55-61 (2010)

In view of the above circumstances, the present invention enhances mucosal immunity efficiently when an antigen is administered to a mucosal tissue (hereinafter referred to as “mucosal administration”), thereby producing an antibody specific to the antigen. It is an object of the present invention to provide a novel adjuvant for mucosal administration that can be enhanced and that is stable and safe when applied to a living body. Another object of the present invention is to provide a composition for enhancing antibody production comprising such an adjuvant and an antigen as active ingredients.

In order to solve the above problems, the present inventor has paid attention to TNF-α, and has conducted extensive research and repeated various trials and errors, so that TNF-α not only lacks stability in a solution state, It was found that there was a problem that when it was administered to mucosa, it was rapidly deactivated and only a weak adjuvant effect could be exhibited.

The present inventor conducted further research to solve such problems, and as a result, a complex of TNF-α and cholesterol pullulan (hereinafter referred to as “TNF-α-cholesterol pullulan complex” or “TNF / CHP”). However, by performing mucosal administration at the same time as the antigen or before and after antigen administration, uncomplexed TNF-α (hereinafter referred to as uncomplexed TNF-α is simply referred to as “ As compared with the case of using “TNF-α”), it has been found that it exhibits a strong adjuvant effect that enhances the production of a specific antibody against an antigen in a living body. Furthermore, the potentiation effect is known as the mucosal administration adjuvant, and the strength of the action effect is known, and when developing the mucosal administration vaccine, it was found to be equal to or greater than the cholera toxin B subunit that is widely used as a positive control. . Furthermore, the present inventor has found that when a composition containing an antigen used for a vaccine or the like and a TNF-α-cholesterol pullulan complex is administered to a living body, particularly when administered intranasally, such antigen-specific IgG And IgA antibody production, in particular, secretory IgA antibody production, and when a component derived from a pathogenic microorganism is used as an antigen, the body to which it is administered is protected against infection against the pathogenic microorganism. It was found that the ability (including cross-protection ability) and / or the ability to prevent serious infection can be effectively imparted.

Furthermore, the present inventor has shown that TNF-α in the state of TNF-α-cholesterol pullulan complex is stable even in the state of an aqueous solution and is used alone or as an antigen or one pharmaceutically acceptable one. It has been found that it is extremely stable even in a preparation containing two or more components.

Furthermore, the present inventor has confirmed that the TNF-α-cholesterol pullulan complex and the preparation containing the same are safe compositions that do not induce serious side effects even when applied to a living body. The invention has been completed.

That is, the present invention provides a mucosal adjuvant containing the TNF-α-cholesterol pullulan complex as an active ingredient, and a composition for enhancing antibody production for mucosal administration containing such an adjuvant and an antigen. The present invention solves the above problems.

In another aspect different from administration to mucosal tissue, the TNF-α-cholesterol pullulan complex of the present invention may be administered subcutaneously, intradermally, intramuscularly or the like simultaneously with the antigen or before and after the antigen administration. By administering to a site other than mucosal tissue by the skin route, it can be used as an adjuvant that effectively enhances the production of antibodies against antigens, particularly IgG antibodies. Intravascular administration is also possible, but when TNF-α-cholesterol pullulan complex is excessively administered intravascularly, if the amount of TNF-α released from the complex into the blood is large, side effects may occur. Therefore, it is necessary to adjust the number of administrations and the dose as appropriate to suppress the rapid increase in the blood concentration of TNF-α. Although administration of antigen to mucosal tissues in body cavities such as the abdominal cavity, thoracic cavity, and middle ear cavity is a percutaneous route using injection, catheter, endoscope, etc., TNF-α-cholesterol pullulan complex When the administration site is mucosal tissue and the production of secretory IgA antibody specific for the administered antigen is enhanced in the mucosal tissue other than the administration site and the administration site, when the administration site is mucosal tissue, the administration route Regardless of the case, in this specification, all are included in mucosal administration (administration to mucosal tissue).

The antigen used together with the adjuvant for mucosal administration comprising the TNF-α-cholesterol pullulan complex of the present invention as an active ingredient is usually used as a vaccine for transdermal administration to humans, for example, influenza, Japanese encephalitis, measles, rubella , Antigens derived from pathogenic microorganisms such as viruses such as yellow fever, lassa and dengue, bacteria such as tuberculosis, protozoa such as malaria, tetanus, diphtheria, pertussis, hemorrhagic colitis, meningitis, etc. And an antigen derived from a substance having cytotoxicity, such as a toxin derived from the causative bacteria of bacterium and amyloid β peptide. Moreover, the antigen derived from pathogenic microorganisms, such as foot-and-mouth disease and Newcastle disease used for the purpose of infection prevention etc. with respect to animals other than a human, can be mentioned.

The adjuvant for mucosal administration of the present invention is the nasal cavity, eye, throat, trachea, bronchi, lung, vagina, oral cavity, digestive tract, and body cavity at the same time as or before and after the antigen is administered to humans or non-human vertebrates. By administering it to mucosal tissues in the inside, it is possible to effectively enhance the production of IgA antibodies and IgG antibodies effective in protecting against pathogenic microorganisms having such antigens and neutralizing toxic substances. .

Further, the composition containing the adjuvant for mucosal administration of the present invention and the antigen is used in human or non-human vertebrates such as mucous membranes in the nasal cavity, eyes, throat, trachea, bronchi, lungs, vagina, oral cavity, digestive tract, and body cavity. By administering to a tissue, it is possible to effectively enhance the production of IgA antibodies and IgG antibodies that are effective in protecting against pathogenic microorganisms having such antigens and neutralizing toxic substances.

Further, the adjuvant for mucosal administration of the present invention is not only against pathogenic microorganisms having antigens administered simultaneously or before or after administration to mucosal tissues, but also against closely related (subtype) pathogenic microorganisms. Cross defense ability can be induced.

Furthermore, the composition containing TNF-α-cholesterol pullulan complex or TNF-α-cholesterol pullulan complex and antigen, which is an active ingredient of the adjuvant for mucosal administration of the present invention, is applied to humans and non-human vertebrates. In this case, since the immune response to the administered antigen can be enhanced by administering a relatively low dose of the antigen, the problem of tolerance of the immune response that may be induced when a large amount of antigen is administered can be avoided. In addition, when it is required to prepare a large amount of vaccine in a short period of time, such as a pre-pandemic vaccine, it can be handled with a smaller amount of antigen than the conventional transdermal vaccine, so a sufficient amount necessary for large-scale vaccination The period for producing the vaccine can be shortened.

Furthermore, the composition containing TNF-α-cholesterol pullulan complex or TNF-α-cholesterol pullulan complex and antigen, which is an adjuvant for mucosal administration of the present invention, can be applied to humans and animals other than humans. It is a safe composition that does not cause inactivation or disappearance in a short time as when TNF-α is administered, is stable, and does not induce serious side effects.

1. Definition of terms:
In this specification, the following terms have the following meanings.

<Adjuvant>
The term “adjuvant” as used herein refers to a substance having a function of enhancing a specific immune response to an antigen by nonspecifically stimulating the immune mechanism.

<Mucosal administration adjuvant>
As used herein, “mucosal administration adjuvant” refers to those having an action of specifically enhancing an immune response to an antigen when administered to a mucosal tissue simultaneously with an antigen or before and after administration of the antigen. And those having the effect of enhancing the production of antigen-specific antibodies in the blood.

<Mucosa tissue>
As used herein, “mucosal tissue” refers to mucosal tissue present on the body surface that is in direct contact with the outside world. Specific examples include the nasal cavity, eyes, throat, trachea, bronchi, lungs, vagina, mucous tissue of the gastrointestinal tract from the oral cavity and esophagus to the large intestine, and further inside the body cavity such as the thoracic cavity, abdominal cavity and inner ear cavity. Including mucosal tissue.

<TNF-α>
Unless otherwise specified, “TNF-α” as used herein refers to a human-derived polypeptide consisting of 157 amino acids and having a molecular weight of about 17.3 kilodaltons (kD) (GenBank Accession Number M16441 and 2251 (refer to the amino acid sequence of wild-type TNF-α represented by SEQ ID NO: 1) in the sequence listing described in the publication No.-2251, and includes both natural and recombinant types. Usually, TNF-α molecules are in the form of trimers under physiological conditions.

<Variant TNF-α>
The term “mutant TNF-α” used herein refers to a polypeptide in which 1 to 6 of the amino acids constituting the TNF-α are deleted or substituted with other amino acids, The biological activity has the same adjuvant activity as wild-type TNF-α. Specifically, the 11th, 65th, 90th, 98th, 112th and 128th lysines from the N-terminal of the amino acid sequence of the wild-type TNF-α peptide disclosed in JP-A-2004-2251 are all included. Examples include peptides substituted with any of asparagine, alanine, arginine, serine, threonine, purine, methionine, or leucine. All of these peptides have an adjuvant activity equal to or greater than that of the wild type.

<Activity of TNF-α (JRU)>
As used herein, “TNF-α activity (JRU)” is based on a Japanese standard product (LotNo. J-PS5K01) supplied by the National Institute of Infectious Diseases (former National Institute of Preventive Health). It is measured by a bioassay using the cytotoxic activity against a blast cell line (mouse LM cell) as an index. The specific activity of pure human TNF-α is approximately 2 × 10 ⁶ JRU / mg protein.

<Cholesterol pullulan>
As used herein, “cholesterol pullulan” refers to a compound in which a cholesteryl group is introduced into a part of the OH group of pullulan of a water-soluble polysaccharide. Such cholesterol pullulan forms monodisperse nanoparticles having a particle size of about 20 to 100 nm by self-association between several molecules using a cholesteryl group as a non-covalent cross-linking point in an aqueous solution. A complex can be formed with a protein (see, for example, Patent Documents 5 and 6). Similarly, it includes pullulan in which an analog of a cholesteryl group in which an alkyl group or the like is further introduced into a cholesteryl group, which can form a nanoparticle and form a complex with a spherical protein. In view of the strength of adjuvant activity as a TNF-α complex, pullulan into which a cholesteryl group is introduced is most desirable.

<TNF-α-cholesterol pullulan complex>
The term “TNF-α-cholesterol pullulan complex” as used herein refers to other than covalent bonds such as hydrophobic bonds in which TNF-α protein is incorporated into cholesterol pullulan nanoparticles formed by self-association in an aqueous solution. A complex formed according to the binding mode. The fact that the binding mode of such a complex is other than a covalent bond is that, as described later, methyl β-cyclodextrin having a high affinity for a cholesteryl group is added to a solution containing TNF-α-cholesterol pullulan. Thus, TNF-α is released, and TNF-α activity that was not observed before the addition of methyl-β-cyclodextrin can be easily confirmed.

<DDS conversion>
The term “DDS conversion” as used herein refers to a dosage form that can control the release of a drug using a DDS technique for precisely controlling the dynamics of the drug in vivo. .

<Antigen>
The term “antigen” as used herein refers to a substance that induces specific antibody production when administered to humans or non-human vertebrates, and has no ability to induce antibody production when administered alone. Also include those that elicit antibody production when administered with an adjuvant. Also included are those capable of inducing antibody-producing ability by binding to substances such as polysaccharides.

2. About the present invention:
As described above, the present invention relates to a mucosal administration adjuvant containing a TNF-α-cholesterol pullulan complex as an active ingredient, and a composition for enhancing antibody production against the antigen containing such a mucosal administration adjuvant and an antigen. It is.

TNF-α itself used in the present invention is a known cytokine, and its use as an adjuvant has also been proposed (Patent Document 3). However, as shown in the Examples described later, it has been found that even if TNF-α is administered to the mucosa together with the antigen, the effect as an adjuvant is not so much exhibited.

The TNF-α used in the present invention may be a natural type, regardless of its origin or origin, and may be a recombinant TNF-α (hereinafter referred to as “rec-TNF-α”) produced by genetically modified DNA technology. May be abbreviated), may be chemically synthesized from amino acids as raw materials, and for example, amino acids constituting TNF-α as disclosed in Non-Patent Document 4 Mutant TNF-α in which a part of the amino acid is substituted with another amino acid may be used. However, particularly when administered to humans, the antigenicity to humans is low, and the risk of reducing or disappearing the adjuvant effect and inducing side effects. Therefore, human wild-type TNF-α is desirable. TNF-α is usually present in trimeric form under physiological conditions. In addition, when used for animals other than humans, TNF-α derived from a target administration animal can also be used.

The pullulan used for the preparation of cholesterol pullulan in the present invention has a mass average molecular weight of 10,000 to 1,000,000 daltons, preferably 50,000 to 500,000 daltons, 75,000 to 250,000 daltons. Things are more desirable. Considering that the TNF-α-cholesterol pullulan complex of the present invention shown below is for pharmaceutical use, it is desirable that the molecular weight is uniform, and the value obtained by dividing the mass average molecular weight by the number average molecular weight is more than 2.5. Small ones are desirable, 2.0 or less are more desirable, and 1.5 or less are particularly desirable.

The pullulan used for the preparation of cholesterol pullulan in the present invention can be prepared by a method such as a fermentation method or a synthesis method regardless of its origin or origin. From the viewpoint of economy, the fermentation method is desirable. Commercially available pullulan (manufactured by Hayashibara Co., Ltd.) may be used. For example, gel filtration chromatography or fractional precipitation described in JP-A-57-141401 is applied to such pullulan as a raw material. However, the molecular weight distribution may be narrowed.

In the cholesterol pullulan used in the present invention, a part of the hydroxyl group of the glycosyl group constituting the pullulan molecule has the following formula: —O— (CH ₂ ) _m CONH (CH ₂ ) _n NH—CO—O—R (wherein R Is a cholesteryl group or an analog thereof; m is 0 or 1; n is an integer of 2 to 6). As such cholesteryl group, 0.5 or more and 10 or less introduced per 100 glycosyl groups constituting the pullulan molecule are used, preferably 1 or more and 3 or less are introduced, and 1 or more and 2 or less are introduced. This is particularly desirable. Incidentally, the amount of cholesteryl group introduced per 100 glucosyl groups of the pullulan molecule can be determined from the proton integration ratio of pullulan and cholesteryl group by ¹ H-NMR measurement.

There is no restriction | limiting in particular in the manufacturing method of the cholesterol pullulan used by this invention, A cholesteryl group thru | or its analog should just be introduce | transduced into a pullulan by a conventional method. Further, for example, commercially available cholesterol pullulan (trade name “PUREBRIGHT CP-100T”, sold by NOF Corporation) can also be used. Incidentally, this commercially available cholesterol pullulan has the following formula: —O—CONH (CH ₂ ) ₆ NH—CO—O— per 100 hydroxyl groups at the 6-position of a glycosyl group constituting a pullulan molecule having a molecular weight of about 100,000 daltons. It has a structure having 1 or more and 2 or less groups represented by R (wherein R is cholesterol).

As described above, cholesterol pullulan itself is a known hydrophobized polysaccharide, and when a cholesterol pullulan-antigen complex is administered to an animal having an antibody-producing ability, an immune response to the antigen is enhanced. The use of pullulan as an adjuvant for vaccines has also been proposed (Patent Documents 5 and 6). However, as shown in the examples below, even if cholesterol pullulan and antigen are mixed and simultaneously administered to mucosa, a complex with antigen is not usually formed in a short time. In the dosage form, it has been found that the effect of cholesterol pullulan itself as an adjuvant is not so great.

Furthermore, since a complex of cholesterol pullulan and an antigen is usually used to enhance an immune response to the antigen (see, for example, Patent Document 5 and Patent Document 6), for example, a complex of TNF-α and cholesterol pullulan is used. When administered, the production of antibodies against TNF-α is enhanced, and there is a concern that the risk of neutralizing the adjuvant activity of TNF-α is increased. However, the present inventors have found that in the case of TNF-α-cholesterol pullulan complex, production of antibody against TNF-α is not induced even if it is administered to mucosal tissue multiple times. It was found that suppression of adjuvant activity did not occur.

The method for preparing the TNF-α-cholesterol pullulan complex according to the present invention is not particularly limited. For example, as the cholesterol pullulan, 100 hydroxyl groups at the 6-position of the glucosyl group constituting the pullulan molecule having a molecular weight of 100,000 daltons. The one having a structure having one or more and two or less groups represented by the following formula: —O—CONH (CH ₂ ) ₆ NH—CO—O—R (wherein R is cholesterol) is used. In this case, the cholesterol pullulan is added to an aqueous solvent such as phosphate buffered saline (PBS) at 50 to 60 ° C. in advance, and the mixture is stirred and mixed. Dissolve completely. To this cholesterol pullulan solution, TNF-α dissolved in an aqueous solvent such as PBS is added, and stirred and mixed to be uniform, and then kept at room temperature to 40 ° C., preferably 25 ° C. to 37 ° C. for 2 days to 7 days. By allowing to stand for 4 days, preferably 4 to 6 days, one TNF-α trimer is usually incorporated into the nanoparticles formed by cholesterol pullulan to form a TNF-α-cholesterol pullulan complex. Is done. The TNF-α-cholesterol pullulan complex thus prepared can be used as an adjuvant as it is. The TNF-α-cholesterol pullulan complex used in the present invention is usually prepared by mixing TNF-α and cholesterol pullulan at a molar ratio of 1: 1 to 30, and more efficiently TNF-α- A ratio of 1: 2 to 20 is desirable for preparing cholesterol pullulan complex, and a ratio of 1: 3 to 15 is particularly desirable. In the case of such a mixing ratio, almost the entire amount of TNF-α can be complexed. If the mixing ratio of TNF-α to cholesterol pullulan is higher than this ratio, the amount of TNF-α increases. Therefore, there is a high possibility that side effects caused by TNF-α will occur depending on the dose when used as an adjuvant. In some cases, a purification step using a gel filtration method or the like is required. Conversely, when the mixing ratio of cholesterol pullulan is higher than this ratio, the proportion of TNF-α-cholesterol pullulan complex in the total nanoparticles decreases, and the proportion of nanoparticles that do not contain unnecessary TNF-α as an adjuvant increases. Since it increases, it is not preferable.

The particle size of the nanoparticles formed by cholesterol pullulan complexed with TNF-α prepared by such a method is determined by a commercially available particle size distribution measuring device (trade name “Shimadzu Laser Diffraction Particle Size Distribution Measuring Device (SALD-7100)”). ”, Manufactured by Shimadzu Corporation), it is about 20-100 nm. The formation of the TNF-α-cholesterol pullulan complex is due to the fact that the peak of TNF-α protein used as a raw material decreased or disappeared by gel filtration chromatography, and a new single molecule was found on the polymer side of cholesterol pullulan. This can be confirmed by the appearance of a peak. In addition, when TNF-α and cholesterol pullulan are mixed at the appropriate ratio to form a complex, most of TNF-α forms a complex. Therefore, when the TNF-α activity of such a solution is measured, Is not detected or only slightly detected. Further, as shown in the examples described later, by adding PBS containing methyl-β-cyclodextrin to the solution, TNF-α is released from the complex, and its activity can be detected. .

There is no particular limitation on the antigen used together with the mucosal administration adjuvant of the present invention, but in particular, the target pathogenic microorganism and / or its target by transdermal administration or mucosal administration in humans or non-human vertebrates to be administered. Antigens that can be confirmed to have the ability to induce antibody production effective against infection against subtype pathogenic microorganisms and can be confirmed to be safe even when administered are desirable. Examples of such antigens include antigens derived from pathogenic microorganisms conventionally used as vaccines for transdermal administration, and antigens derived from allergens that cause allergies used in allergy desensitization therapy. be able to. More specifically, vaccines such as inactivated vaccines, live vaccines, component (component) vaccines, multivalent vaccines, mixed vaccines, and DNA recombinant vaccines, and antibody production that neutralizes cytotoxins produced by pathogenic microorganisms Antigens such as toxoids that are used for the purpose of induction of the disease are desirable, and vaccines for protecting against infection of pathogenic microorganisms that are infected by the mucosal tissue route according to the properties of the adjuvant of the present invention, which is excellent in the secretory IgA antibody production enhancing action in the mucosal tissue The antigen used as is particularly desirable. More specifically, for example, when applied to humans, respiratory diseases such as influenza, cold, pneumonia, wind shin, measles, otafukaze, yellow fever, dengue fever, Lassa fever, Japanese encephalitis, polio, chickenpox, AIDS, severe acute Respiratory syndrome (SARS), hepatitis such as hepatitis A, hepatitis B, hepatitis C, herpes, cervical cancer, pneumonia, rabies, antigens derived from viruses such as diarrhea and other diseases, tuberculosis Bacteria, Shigella, Cholera, Salmonella typhi, Pest, Haemophilus influenzae, Streptococcus mutans, Polyphyromonas gingivalis, Porphyromonas pathogen Bacteria and ma Pathogenic protozoa, such as the rear, and the like outer membrane proteins such as mycoplasma. In particular, surface proteins involved in infection are mutated every year, such as influenza viruses, and existing transdermal vaccines are unable or insufficient to induce blood IgG antibody production that is effective in protecting against infection. It is useful as a pre-pandemic vaccine for rapidly inducing infection-protective ability based on cross-protection reaction against pathogenic microorganisms that cause a global pandemic. In addition, if it has antigenicity, not only complex proteins such as glycoproteins, but also nucleic acids, polysaccharides, lipids, etc., and partial degradation products containing epitopes that have the ability to induce antibody production effective for infection protection should be used. You can also. More specifically, the inactivated vaccine includes, for example, cholera vaccine, pertussis diphtheria mixed vaccine, pertussis diphtheria tetanus mixed vaccine, Weil disease autumn gonorrhea mixed vaccine, influenza vaccine, Japanese encephalitis vaccine, dry Japanese encephalitis vaccine, inactivated rabies disease Examples of the vaccine include dry tissue culture inactivated rabies vaccine. Moreover, as a live vaccine, a pressure ulcer vaccine, a dry pressure ulcer vaccine, a dry cell culture pressure ulcer vaccine, an oral live polio vaccine, a dry attenuated live wind shin vaccine, a dry attenuated otafukase vaccine can be illustrated, for example. Japanese encephalitis vaccine, measles vaccine, rubella vaccine, mycoplasma vaccine, papillomavirus vaccine, rotavirus vaccine and toxoids include, for example, diphtheria toxoid, adult precipitated diphtheria toxoid, precipitated tetanus toxoid, diphtheria tetanus mixed toxoid, precipitated diphtheria tetanus Examples thereof include mixed toxoids and precipitated hub toxoids. These are all antigens and toxoids that are usually used as vaccines for protection against infection and / or prevention of the onset and severity of clinical symptoms due to infected pathogenic microorganisms. Furthermore, it may be a protein or peptide having cytotoxicity such as a toxin causing hemorrhagic colitis such as verotoxin or amyloid β peptide. Examples of allergens include pollen-derived allergens such as cedar and ragweed and mites.

Examples of antigens to be applied when applied to animals other than humans include antigens derived from pathogenic microorganisms such as foot-and-mouth disease virus, Newcastle disease virus, influenza virus that infects various animals, rabies virus, and anthrax be able to. Examples of antigens other than those described above include enzymes, cell surface markers, tumor markers, antibodies and the like used as reagents and clinical diagnostic agents.

Further, an antigenic determinant (epitope) portion in an antigen or a substance containing the same may be used. Furthermore, when the antigen is a protein or peptide, it is disclosed in, for example, International Publication No. 2004/87767 pamphlet. Synthetic peptides capable of inducing antibody production against B cell epitopes by artificially linking such T cell epitopes having the ability to induce immune responses and B cell epitopes of antigens intended for antibody production, Synthetic peptides in which a B cell epitope is linked to a carrier molecule as disclosed in Japanese Patent No. 515006 / may be used. In addition, even if the substance itself is a substance that does not have the ability to induce antibody production even when administered alone, it can induce antibody production when used in combination with the TNF-α-cholesterol pullulan complex of the present invention. Needless to say, those that have been combined with a carrier molecule or the like to impart antigenicity, or those that can induce antibody production when used in combination with other adjuvants can also be used.

The antigen used together with the adjuvant for mucosal administration of the present invention is not limited in its production method or origin, and microorganisms expressing the antigen and products containing the antigen produced by the microorganism can be used. It is also possible to use those produced by chemical synthesis or those chemically synthesized. When the target antigen itself is toxic to the living body, such as tetanus, diphtheria toxin, amyloid-β peptide, or when it is necessary to use infectious microorganisms such as polio, these are usually used. Detoxified or attenuated or peptides having a partial sequence thereof can also be used. These antigens may be used as a crude extract, a partially purified product, or a highly purified product as long as it does not cause serious damage when administered to a living body. For the purpose of producing antibodies against only a specific antigen, or from the viewpoint of reducing the occurrence of side effects, it is desirable to use a purified product of the target antigen as highly as possible. In addition, in the case of a component that does not have a low ability to induce antibody production when administered alone, as described above, a T cell epitope linked or bound to a carrier molecule or the like to enhance antigenicity is used. In addition, an adjuvant other than the TNF-α-cholesterol pullulan complex can be used in combination. In addition, the antigen used for the production of a commercially available vaccine preparation may be used as it is. In particular, when an inactivated vaccine or a live attenuated vaccine is used as an antigen, it is desirable to use it as it is.

The administration route in the case of separately administering the mucosal administration adjuvant and antigen according to the present invention may be either a mucosal tissue route or a transdermal route, but for pathogenic microorganisms whose infection route is mucosal tissue, In order to induce the production of secretory IgA antibody particularly effective for the protection of infection, it is desirable to administer via mucosal tissue, and in view of ease and certainty of administration, mucosal administration into the nasal cavity or oral cavity is desirable. Intranasal administration is more desirable. In order to enhance the production of blood IgG antibody or IgA antibody, administration by mucosal tissue or transdermal route such as subcutaneous, intradermal, intramuscular can be selected depending on the situation. In this case, the dose of the antigen is not particularly limited as long as it is a dose that can enhance antibody production to the living body by using the TNF-α-cholesterol pullulan complex of the present invention in combination. Depending on the strength, it may be adjusted as appropriate. In the case of mucosal administration, usually, 0.01 μg to 100 μg, preferably 0.1 μg to 10 μg, may be administered as an antigen mass per adult with a body weight of 50 kg. When the dose of antigen is less than 0.01 μg, antibody production may not be induced, and even when doses exceeding 100 μg are administered, an effect commensurate with the dose may not be observed. In addition, when the administration target is a vertebrate other than a human, the dosage may be appropriately adjusted in consideration of the volume of the administration site based on the dosage to human.

There are no limitations on the method of antigen used when mixed with the mucosal administration adjuvant according to the present invention, as long as it is a method that can enhance the production of a specific antibody against the antigen. It may be appropriately determined in consideration of the administration route, administration method, animal to be administered and the like. Usually, when an antigen used in an existing vaccine is used, once to several times a day to once a week to What is necessary is just to administer about 1 to 5 times at intervals of about once a month. Usually, sensitization necessary for protection against infection is established by a single administration, but when used in the epidemic of highly contagious pathogenic microorganisms such as influenza virus, or antigenicity has been mutated to cause a pandemic. When it is necessary to effectively enhance the cross-protection ability in a short period of time, as in the case of subtype pathogenic microorganisms, once to several times a day, once a week to once a month It may be administered 2 to 5 times, preferably 2 to 4 times, more preferably 3 or 4 times. The dosage in the case of using other antigens may be appropriately adjusted while confirming the presence / absence of the production of antigen-specific antibodies and the production amount thereof according to the above or by an appropriate method. In addition, when administered by a transdermal route to a site other than mucosal tissue, an inflammatory reaction may be induced at the site of administration depending on the dose. The amount needs to be reduced accordingly.

The adjuvant for mucosal administration of the present invention, when administering an antigen to a human or an animal by mucosal administration or other site by a transdermal route, before or after administration of the antigen, more preferably simultaneously with administration of the antigen, Even when administered to a mucosal tissue other than the administration site of the antigen, or transdermally administered to other sites, it can exert an excellent adjuvant effect, but the enhancement effect of antibody production is strong From this point, it is desirable to administer to the antigen administration site. From the viewpoint of enhancing production of secretory IgA antibody, ease of administration, and reducing the risk of inducing side effects, mucosal administration is desirable, nasal or buccal administration is desirable, and nasal administration is particularly desirable. When administered separately from the antigen, it is preferably administered within 0 to 4 hours before administration of the antigen, more preferably within 0 to 2 hours, and particularly preferably at the same time as possible. It is possible to administer after antigen administration, but in this case as well, it is necessary to administer as soon as possible after antigen administration, preferably within 0 to 3 hours. Adjuvant effect may not be fully exerted if administration is carried out after an antigen administration for a long time.

The mucosal administration adjuvant of the present invention is provided in the form of a composition containing TNF-α-cholesterol pullulan complex as an active ingredient, or in the form of a composition mixed with an antigen for the purpose of enhancing antibody production. . Moreover, it is good also as a form of the composition which mixed the appropriate quantity with the antigen at the time of use.

As long as the effect of the present invention is not hindered, the mucosal administration adjuvant according to the present invention or the composition containing the mucosal administration adjuvant and antigen is used for one or more pharmaceutically acceptable formulations other than these components. Pharmaceutical compositions can also be prepared by combining additives. Examples of pharmaceutical additives include water, physiological saline, phosphate buffered saline (PBS), solvents such as alcohol, reducing carbohydrates such as glucose and maltose, α, α-trehalose, sucrose, and cyclos. Non-reducing carbohydrates such as dextrin, or α, α-trehalose carbohydrate derivatives such as α-glucosyl α, α-trehalose, α-maltosyl α, α-trehalose, sorbitol, mannitol, maltitol, maltotriitol Sugar alcohols such as agar, pullulan, guar gum, gum arabic, methyl cellulose, polyvinylpyrrolidone and other water-soluble polymers, lipids, amino acids such as sodium L-glutamate and salts thereof, buffers, stabilizers, antibacterial agents, absorption promotion Agent, surfactant, preservative, antioxidant, solubilizer, pH adjuster, fragrance, nutritional functional food, Examples include quasi-drugs or active ingredients of pharmaceuticals, foods other than those mentioned above, food additives, quasi-drug additives, pharmaceutical additives, etc., and one or more of these may be used in appropriate combination Can do. If necessary, an adjuvant other than TNF-α-cholesterol pullulan according to the present invention can be blended. Of these, α, α-trehalose and α, α-trehalose saccharide derivatives, which have a high stabilizing effect on TNF-α and other additives, are preferred. It should be noted that addition of a component having a high affinity for a cholesteryl group, such as a protein other than an antigen or a carbohydrate such as methyl β-cyclodextrin, releases TNF-α from the TNF-α-cholesterol pullulan complex. It is not preferable as an additive.

Incidentally, in the case of the composition containing the adjuvant for mucosal administration and the antigen according to the present invention, since the antigen is often a protein or peptide, the TNF-α-cholesterol pullulan complex resulting from the large amount of the antigen at the time of preparation. The concentration of the antigen mixed with the TNF-α-coreterol pullulan complex needs to be as low as possible so that the release of TNF-α from the mixture does not occur. Usually, the antigen concentration in the composition is 10 mg / ml or less. What is necessary is just to mix so that it may become 5 mg / ml or less, and it is more desirable to set it as 1 mg / ml or less.

The mucosal administration adjuvant of the present invention or the composition containing such a mucosal administration adjuvant and an antigen includes solutions, syrups, freeze-dried products, powders, granules, tablets, troches, sublingual tablets, creams. From the dosage forms such as ointments and gels, it may be appropriately selected in consideration of the administration subject, administration method, storage method of the preparation and transport method. Further, the mucosal administration adjuvant according to the present invention or the composition containing the same can be penetrated into the site where the antigen-presenting cells exist by using a penetration enhancer or iontophoresis method into the skin or tissue as necessary. Can also be promoted. Moreover, the formulation which concerns on this invention can also be administered transmucosally by making it into the form of various food / beverage products, such as tablet confectionery, a candy, and a soft drink, if it is taken orally.

There is no particular limitation on the method of administering the pharmaceutical composition containing the adjuvant for mucosal administration according to the present invention as an active ingredient, or the pharmaceutical composition containing such an adjuvant for mucosal administration and an antigen as active ingredients, Any method may be used as long as the mucosal administration adjuvant according to the present invention or the composition containing the adjuvant and the antigen as active ingredients can reliably reach the administration site. In the case of mucosal administration, for example, an appropriate amount may be dropped on the mucous membrane using a syringe or syringe, orally taken, applied to the mucosa in the form of a cream or gel, or guided to the administration site with a catheter or the like. Moreover, it may be sprayed in the form of a mist with a spray or nebulizer on the nose or throat, or may be aspirated into the trachea, bronchus or lung, or administered into the large intestine in the form of a suppository. In addition, for administration into body cavities such as subcutaneous, intradermal, intramuscular, intravascular, intraperitoneal and intrathoracic, use an appropriate administration method depending on the administration site and administration route, such as a syringe, catheter, infusion, etc. Can do.

The composition containing the adjuvant for mucosal administration of the present invention as an active ingredient or the composition for enhancing antibody production containing such an adjuvant for mucosal administration and an antigen as active ingredients is one or more other pharmaceutical compositions It can also be used simultaneously or sequentially. After administering another pharmaceutical composition, a pharmaceutical composition containing the mucosal administration adjuvant according to the present invention as an active ingredient may be administered, and after administering the pharmaceutical composition, another pharmaceutical composition may be administered. In addition, the pharmaceutical composition and the chemotherapeutic agent may be administered simultaneously. Other pharmaceutical compositions vary depending on the target disease, but examples include other vaccines, chemotherapeutic agents, antibody drugs, antisense nucleic acid drugs, siRNA drugs, and the like.

The use of the adjuvant for mucosal administration according to the present invention may be determined as appropriate in consideration of the ability to induce antibody production of the antigen, the type of disease, administration route, administration method, animal to be administered, etc. Administered. Specifically, for example, when using an antigen used in an existing vaccine, the usage of the vaccine may be followed, usually once to several times a day, preferably at intervals of 1 to 30 days, preferably The administration may be performed 1 to 14 days, particularly preferably 1 to 14 days, and 1 to 5 times, preferably 2 to 4 times. When using with antigens other than vaccines, according to the above or depending on the antigenic strength of the antigen to be used, while confirming the presence or absence of antigen-specific antibody production and the amount of antibody production by an appropriate method, What is necessary is just to adjust suitably. In the case of mucosal administration, the dosage is 0.1 to 5,000 μg / time, preferably 0.1 to 2,500 μg / time as TNF-α protein for an adult weighing 50 kg as TNF-α protein. Multiple doses, more preferably 0.1-100 μg / dose. Depending on the immunogenicity of the antigen used and the dose, the adjuvant effect may not be exhibited at doses less than 0.1 μg / dose, and even if doses exceeding 5,000 μg / dose are administered, the dose will be commensurate with the dose. Adjuvant effect may not be obtained. In addition, when the administration target is a vertebrate other than a human, the dosage may be appropriately adjusted in consideration of the volume in the nasal cavity based on the dosage to human.

The adjuvant of the present invention is highly versatile and can be administered to mucosal tissues and blood in the whole body such as saliva and nasal fluid by administration to mucosa such as nasal administration together with various antigens including antigens used as vaccines. Furthermore, the production of antibodies specific to the administered antigen, in particular, secretory IgA antibodies, is significantly enhanced. Since the secretory IgA antibody has a high cross-protective ability, it is extremely effective as an adjuvant for enhancing antibody production against an antigen for mucosal administration, and can induce the cross-protective ability. When used in combination, the organism can protect against such microorganisms and closely related (subtype) microorganisms and / or develop diseases caused by microbial infection (including diseases caused by toxic substances derived from microorganisms) Severe prevention can be efficiently imparted. Moreover, in the case of mucosal administration, the risk of inducing antibody production against TNF-α itself is extremely low. When the adjuvant of the present invention is administered together with the antigen to a site other than the mucosal tissue by a transdermal route, the IgG and / or IgM antibody specific to the administered antigen and the production of IgA antibody are produced in the blood. Therefore, the adjuvant of the present invention is extremely effective as an adjuvant for enhancing antibody production against various antigens administered by a transdermal route to a site other than mucosal tissue.

The TNF-α-cholesterol pullulan complex of the present invention is not only useful as an adjuvant, but can maintain sustained release even when administered by a transdermal route to a site other than a mucosal tissue. Compared to the administration of α, the same effect can be achieved at a low dose and the occurrence of side effects can be reduced. Therefore, breast cancer, liver cancer, renal cancer alone or in combination with other therapeutic agents For the treatment of TNF-α-sensitive diseases such as malignant tumors and viral diseases such as melanoma and mycosis fungoides, and also as a biological preparation for improving vascular permeability Can do.

Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

<Preparation of various DNF-modified TNF-α preparations>
The present inventor has shown through preliminary experiments that even if TNF-α dissolved in physiological saline is administered to mucosal tissues in the nasal cavity, it exhibits only a weak adjuvant effect. In order to investigate the cause, TNF-α dissolved in physiological saline was used as an adjuvant, and it was found that TNF-α was deactivated in a relatively short time when dissolved in physiological saline. . Furthermore, in another experiment, when the present inventor dissolved TNF-α labeled with a fluorescent dye in physiological saline and administered it subcutaneously to mice, rapid disappearance of the fluorescent dye from the administration site was observed. From this, it was also found that TNF-α may diffuse into the tissue quickly from the administration site. From these preliminary experiments, it was suggested that even when TNF-α was administered intranasally, TNF-α was inactivated or disappeared in a short time at the administration site, so that sufficient adjuvant activity could not be exhibited. . Therefore, in order to stably hold TNF-α on the mucosal tissue for a long period of time during mucosal administration, studies were made on its DDS (drug delivery system) or stabilization method. That is, for the DDS conversion or stabilization of TNF-α, a complex with cholesterol pullulan was prepared by the following method. Further, as a control, adsorption to a bioabsorbable hydrogel, which is widely used as a DDS or stabilizer, encapsulation in a subcutaneously absorbable lyotropic liquid crystal (Nanocube), and encapsulation in a liposome were performed. Furthermore, TNF-α was converted to DDS by randomly modifying the amino group of the TNF-α molecule using water-soluble high-molecular polyethylene glycol (hereinafter referred to as “PEG”). In the following examples, natural human TNF-α produced by human lymphoblastoid cell line BALL-1 cells using Sendai virus as an inducer and purified by a conventional method (manufactured by Hayashibara Biochemical Laboratories, Inc., Specific activity of about 2 × 10 ⁶ JRU / mg protein) was used.

<Preparation of TNF-α-cholesterol pullulan complex preparation>
Cholesterol pullulan (trade name “PUREBRIGHT CP-100T”, sold by NOF Corporation) weighs an appropriate amount, and then Dulbecco ’s PBS (trade name “Dulbecco PBS (−) powder“ Nissui ””, Nissui to 20 mg / ml. Suspended in Pharmaceutical Co., Ltd. (hereinafter referred to as “D-PBS”), dissolved in a warm water bath at 55 ° C. for 16 hours, and then sterilized by filtration using a membrane filter having a pore size of 0.22 μm. After mixing 7 ml of this cholesterol pullulan solution and 2 ml of TNF-α (TNF-α protein amount 1.454 mg / ml) dissolved in D-PBS (−), the mixture was sterilized by filtration using a membrane filter having a pore size of 0.22 μm. By reacting at 37 ° C. for 5 days, a TNF-α-cholesterol pullulan complex preparation was prepared. When the TNF-α activity of this solution was measured by a bioassay based on cytotoxic activity using mouse LM cells, no activity was detected. In addition, an equal amount of D-PBS containing 200 mg / ml methyl-β-cyclodextrin was mixed with this solution and reacted at 37 ° C. for 2 hours to release TNF-α out of the cholesterol pullulan nanogel. When diluted with MEM medium containing 1% by volume of fetal serum (FCS) (hereinafter referred to as “MEM medium containing 1% by volume FCS”), the activity was measured in the same manner. Almost the same amount of activity was confirmed.

<Preparation of TNF-α bioabsorbable hydrogel inclusion preparation>
Sheets of two types of commercially available gelatin-based bioabsorbable hydrogels (trade names “MedGel” PI5 and PI9, sold by Medgel Co., Ltd.) used for DDS conversion of physiologically active substances are about 4 mm × about 12 mm each ( The weight was measured with a microbalance and placed in a polypropylene tube (trade name “Falcon 2059 tube”, sold by Becton Dickinson). TNF-α (1.454 mg / ml) was dripped onto a sheet at 20 μl / gel (30 μg / gel as TNF-α protein) and allowed to stand at 4 ° C. overnight. Was prepared.

<Preparation of TNF-α subcutaneously absorbable lyotropic liquid crystal inclusion body preparation>
30 ml of TNF-α (manufactured by Hayashibara Biochemical Laboratories, Inc., 7.54 mg / ml TNF-α protein) and 39.57 μl of glycerin dissolved in PBS are added to a 1.5 ml polypropylene tube (trade name “Eppendorf tube”) , Sold by Eppendorf), and vigorously stirred and mixed using a vortex mixer. Furthermore, 49.26 mg of polyoxyethylene (20) octyldodecyl ether (trade name “Emulgen 2020G-HA”, HLB13, sold by Kao Corporation) was added, and the mixture was vigorously stirred and mixed using a vortex mixer, and then 49.02 μl of squalane. Was added, and vigorously stirred and mixed using a vortex mixer to prepare a TNF-α lyotropic liquid crystal inclusion body. Incidentally, the subcutaneously absorbable lyotropic liquid crystal inclusion body is used as a gel base for cosmetics having a transdermal absorption promoting action (for example, Yamaguchi Y. et al., “Pharmacizie”, Vol. 61, pages 112-116 (2006). Year)).

<Preparation of liposome-encapsulated TNF-α preparation>
TNF-α (manufactured by Hayashibara Biochemical Laboratories, Inc., TNF-α protein 1.454 mg / ml) was diluted with PBS not containing potassium (PBS (K-)), and the concentration of TNF-α protein was 363.5 μg. 2 ml each of an aqueous solution having a concentration of 632 μg / ml and 632 μg / ml was prepared. These solutions are filled into a syringe, and each is a commercially available vial with freeze-dried hollow liposome powder (trade name “COATSOME EL-01-N”, as lipid, L-α-dipalmitoylphosphatidylcholine (DPPC) 54 μmol, cholesterol 40 μmol, 6 μmol of L-α-dipalmitoylphosphatidylglycerol (DPPG) was added, and sold to NOF Corporation. The vial was shaken 3 to 5 times to prepare a liposome-encapsulated TNF-α-containing solution. This solution was subjected to gel filtration column chromatography (trade name “Sephacryl-S300HR”, column size: φ15 mm × 500 mm, sold by Regent Concentrate Co., Ltd.), eluted with PBS (K−), and the amount of protein in the eluted fraction was determined. Using the commercially available Bradford reagent (trade name “Protein Assay Dye”, sold by Bio-Rad Co., Ltd.) and cholesterol detection reagent (trade name “Cholesterol Test Wako”, sold by Wako Pure Chemical Industries, Ltd.) The free TNF-α fraction (protein-containing fraction) and the liposome fraction (cholesterol-containing fraction) were each collected. 0.04 ml of a 1.05 mass% Triton-X100 solution was added to 0.1 ml of the liposome-encapsulated TNF-α-containing solution subjected to the gel filtration column chromatography and 0.1 ml of the liposome fraction eluted from the column, respectively. After 30 minutes of shaking, the liposomes were broken to release the encapsulated TNF-α, and then 1% by volume fetal calf serum (FCS) -containing MEM medium (trade name “Eagle MEM medium“ Nissui ”, code number 05902, The titer of TNF-α in each solution was measured by a bioassay based on cytotoxic activity against mouse LM cells after dilution with kanamycin and phenol red (not available from Nissui Pharmaceutical Co., Ltd.).

<Preparation of PEG-modified TNF-α preparation>
1.67 mg of TNF-α (1.11 mg / ml, 1.98 × 10 ⁶ JRU / ml) in PBS and PEG reagent (method-PEG-Succinimidylpropionate; trade name “MPEG-SPA-20k”, sold by Nectar) ) 6.68 mg was mixed and slowly stirred in a 37 ° C. water bath for 15 minutes. Furthermore, 6.68 mg of PEG reagent was added, and the mixture was slowly stirred and mixed in a 37 ° C. water bath for 15 minutes. In order to stop the reaction, 0.876 mg of ε-aminocaproic acid (10-fold mol of MPEG-SPA-20k) was added, and the mixture was slowly stirred for 10 minutes in a 37 ° C. water bath. The solution after stopping the reaction solution is subjected to gel filtration chromatography (trade name “Superdex200”, column size φ16 mm × 600 mm, gel volume 120 ml, sold by GE Healthcare Co., Ltd.), and the molecular weight per molecule of TNF-α is 20 kilodaltons. Fractions from which molecules having a molecular weight considered to have added 1 to 3 of PEG were eluted were collected to prepare PEG-modified TNF-α. From the elution pattern of gel filtration chromatography, most of the PEG-modified TNF-α prepared and recovered by this method was judged to have PEG1 molecules bound to TNF-α1 molecules.

The DDS conversion rate (mass%) of TNF-α in the preparation prepared by DDS conversion of the five types of TNF-α prepared above, and a half of the DDS-converted TNF-α protein are released in physiological saline. Time (excluding encapsulated TNF-α and PEG-modified TNF-α in liposomes) and DDS-modified TNF-α in a polypropylene tube with a lid under constant temperature conditions of 4 ° C. or 25 ° C. The storage stability when stored is summarized in Table 1. The DDS conversion rate was obtained by dividing the amount of TNF-α protein in the preparation DDS converted by Bradford method by the amount of TNF-α protein used for DDS conversion and multiplying by 100. The deactivation rate (%) at the time of DDS conversion was determined by adding TDS-α converted to DDS, methyl-β-cyclodextrin in the case of TNF-α-cholesterol pullulan complex, and physiological saline in the case of lyotropic liquid crystals. In the case of encapsulation in liposomes, an emulsifier is added and TNF-α is released. Then, the physiological activity and the protein amount are measured by the Bradford method, the physiological activity is divided by the protein amount, and the released TNF is released. Specific activity of -α (JRU / mg protein) was determined. This specific activity was calculated by dividing by the specific activity of TNF-α (2 × 10 ⁶ JRU / mg protein) in the absence of inactivation, multiplying by 100, and subtracting from 100. The PEG-modified TNF-α preparation was similarly calculated based on the results of directly measuring its physiological activity and protein amount. In this case, when there is no deactivation of TNF-α at the time of DDS conversion, the deactivation rate is 0%. The storage stability is determined by storing the DDS sample at the temperature conditions shown in Table 1, sampling every 7 days, measuring the TNF-α activity, and measuring the activity measured for each sample immediately after the DDS conversion to 100. %, The period during which the activity is maintained at 90% or more. In addition, since PEG-modified TNF-α is covalently bonded to PEG and TNF-α, TNF-α is not released, so a release time measurement test was not performed. In addition, since most of TNF-α was inactivated when encapsulated in liposomes, the release time measurement test was not performed. Furthermore, in the adsorption to the hydrogel, the total amount of the added TNF-α solution was absorbed by the gel, so that the DDS conversion rate seemed to be 100%, but the DDS-converted TNF-α protein was 2 min. The time until one amount is released in physiological saline is 0.5 hours or less. Actually, TNF-α was not DDS-converted. Also, TNF-α was not included in liposome encapsulation and PEG modification. Most of α was inactivated. Therefore, all of these methods were judged to be unsuitable for TDS-α conversion to DDS, and no storage stability test was conducted.

As is apparent from Table 1, in the method of forming a complex of TNF-α and cholesterol pullulan, almost the entire amount of TNF-α protein becomes a complex, and the inactivation of TNF-α at the time of complex formation is also physiological. There was no release of TNF-α protein in saline, and its storage stability was excellent (Test Group 1). In contrast, when adsorbed to the hydrogel, almost all of the TNF-α protein used was adsorbed, but TNF-α was released from the gel in a short time in the physiological saline (Test Group 2). When encapsulated in lyotropic liquid crystal, almost the entire amount of TNF-α was converted to DDS, and there was no inactivation during conversion to DDS, and the storage stability was excellent (Test Group 3). When encapsulated in lyotropic liquid crystal, TNF-α was released in a short time because the liquid crystal was easily dissolved in physiological saline. In the encapsulating method in liposomes, the amount of TNF-α to be encapsulated was as extremely low as 2% or less of the TNF-α protein used, and about 60% by mass of TNF-α was inactivated at the time of encapsulation (test group). 4). In the case of modification with PEG, one molecule or more of the TNF-α protein used was modified with PEG, but the inactivation rate of TNF-α was 90% or more, and cells per TNF-α protein amount. The disorder activity (specific activity) was reduced to 1/10 or less compared to TNF-α before PEG modification (Test Group 5). From this result, since TNF-α was not adsorbed to the gel by adsorption to the hydrogel, it was judged unsuitable as a DDS conversion method for TNF-α. In addition, the efficiency of encapsulating in liposomes was extremely low, and TNF-α was inactivated at the time of encapsulating. Therefore, it was judged unsuitable as a DDS conversion method for TNF-α.

Incidentally, the present inventor confirmed by another experiment that PBS containing methyl-β-cyclodextrin at a concentration of 200 mg / ml was used to release TNF-α encapsulated in cholesterol pullulan complex from the complex. For example, 12 mg / ml in the case of interferon-α-cholesterol pullulan complex and about 20 times the concentration in the case of TNF-α-cholesterol pullulan complex. Interferon-α can be easily released by mixing an equal amount with PBS containing -β-cyclodextrin. In addition, the TNF-α-cholesterol pullulan complex prepared above is about 5% in order to release 50% of TNF-α from the TNF-α-cholesterol pullulan complex in a high concentration protein solution such as serum. In the case of interferon-α-cholesterol pullulan complex or interferon-γ-cholesterol pullulan complex, the interferon is completely released within an extremely short time within 30 minutes, and TNF-α Since the DDS effect as in the case of the complex was not observed, the DDS conversion of the cytokine by complexing with cholesterol pullulan has selectivity for the cytokine used, and TNF-α is a cholesterol pullulan complex. It was found that this is a suitable cytokine for DDS conversion.

Using a mouse widely used as a model for confirming the antibody production enhancement effect when an antigen such as a vaccine is administered to humans, the DDS-modified TNF-α prepared in Example 1 is administered to the mucosa simultaneously with the antigen. The effect on antigen-specific antibody production was investigated. As an infection model system for such pathogenic microorganisms to humans, assuming infection protection in the nasal mucosa and upper respiratory tract mucosa as typical natural infection routes of pathogenic microorganisms, representative pathogenic microorganisms of such infection path As an influenza virus selected.

<Antigen>
As an antigen, commercially available influenza HA vaccine (trade name “influenza HA vaccine”, as production strains, A / Brisbane / 59/2007 (H1N1) (A USSR type), A / Uruguay / 716/2007 (H3N2) strain (A Hong Kong type), a preparation using B / Brisbane / 60/2008 strain, sold by Denka Seken Co., Ltd.) and diluted with physiological saline.

<Test method>
<Nasal administration>
Seventy seven BALB / c mice (Charles River Japan, female, 8 weeks old) were randomly divided into 11 groups of 7 mice, and after 1 week of pre-breeding, each of the 5 groups had 7 mice Influenza HA vaccine used (hereinafter sometimes referred to as “influenza vaccine”), TNF-α-cholesterol pullulan complex preparation prepared in Example 1 used as an adjuvant, TNF-α lyotropic liquid crystal inclusion body sample And PEG-modified TNF-α preparation (Test Groups 5, 7, and 8). As a control 1, only physiological saline was administered (test group 1). As a control 2, physiological saline containing influenza vaccine was administered (test group 2). As a control 3, influenza vaccine and TNF-α not treated with DDS were administered as adjuvants (Study Group 3). As a positive control, cholera toxin B subunit confirmed to have an influenza vaccine and mucosal adjuvant activity (sold by Wako Pure Chemical Industries, Ltd., lot number CDH6462 (hereinafter, cholera toxin B subunit may be abbreviated as “CTB” in some cases). (Test group 9) The administration was performed by mixing the adjuvant and the influenza vaccine so that the TNF-α protein, the cholera toxin B subunit and the influenza vaccine protein were in the dosages shown in Table 2, The administration schedule was once a week for 4 weeks (4 administrations), and 15 μl each was administered into the right and left nasal cavities (total 30 μl / animal / dose). The control was mixed with saline and administered, combined with rec-TNF-α and this. Rec-TNF-α was used to prepare rec-TNF-α-cholesterol pullulan complex under the same reaction conditions as the preparation of TNF-α-cholesterol pullulan complex in Example 1, and nasally administered using these preparations The test for confirming the adjuvant activity was carried out (Test group 4 and Test group 6).

Rec-TNF-α is a DNA encoding wild-type TNF-α (see, for example, the DNA sequence of wild-type TNF-α represented by SEQ ID NO: 1 in the sequence listing described in JP-A No. 2004-2251) Is introduced into a commercially available plasmid vector, this plasmid is introduced into Escherichia coli (BL21DE3 strain) according to a conventional method, the resulting transformant is cultured, and the culture is subjected to affinity chromatography, ion exchange chromatography, gel What was purified using filtration chromatography or the like (manufactured by Hayashibara Biochemical Laboratories, Inc., specific activity of about 2 × 10 ⁶ JRU / mg protein) was used.

<Subcutaneous administration>
14 BALB / c mice (Nippon Charles River Co., Ltd., female, 8 weeks old) were randomly divided into 2 groups of 7 mice, and after 1 week of preliminary breeding, As an influenza vaccine (test group 10). The remaining 7 mice per group were administered adjuvant TNF-α-cholesterol pullulan complex and influenza vaccine (Study Group 11). For administration, the adjuvant and influenza vaccine were mixed so that the TNF-α protein and the influenza vaccine protein were in the dosages shown in Table 2, and the 2nd and 4th weeks after the start of breeding (the first time in the case of nasal administration) And 100 μl / animal / dose subcutaneously in the back of the mice (study group 11) in the same administration schedule as the third administration, twice the number of administrations). As a control, physiological saline and influenza vaccine were mixed so that the dosage shown in Table 2 was obtained, and the second and fourth weeks after the start of breeding (the same administration as the first and third administration in the case of nasal administration) 100 μl / animal / dose was administered subcutaneously to the back of the mice (study group 10). From the results of Example 1, among the TNF-α converted to DDS, it was not converted to DDS by adsorption to hydrogel, and the encapsulation in liposome had a very high DDS conversion rate (encapsulation rate in liposome). Since it was found that TNF-α was inactivated, it was considered difficult to convert TNF-α into DDS by these methods, and the adjuvant action was not tested. In addition, when encapsulated in lyotropic liquid crystals, TNF-α is encapsulated in lyotropic liquid crystals, unlike adsorption to hydrogel, and in mucosal administration that can be administered without mixing with an aqueous solvent, the liquid crystals are in mucosal tissue. Since the ratio of contact with body fluid (mucus) is low above, it was considered that TNF-α could be retained at the site of administration for a relatively long period of time, and was subjected to an adjuvant action test.

After 4 weeks of nasal administration or 1 week after the end of the second subcutaneous administration, mice were anesthetized with ether and blood and nasal washes were collected in the usual manner. The amount of IgA antibody specific for the contained influenza vaccine and Enzyme antibody method (EIA) based on HRPO color development reaction using influenza vaccine protein as solid phase, horseradish peroxidase (HRPO) -labeled anti-mouse IgG antibody or IgA antibody, and orthophenylenediamine as color development reagent ). Influenza vaccine protein-specific IgG antibody (hereinafter sometimes referred to as “blood IgG antibody”) in the serum of mice of test groups 1 to 9 and influenza vaccine protein-specific IgA antibody in the nasal lavage fluid (antibody titer) : U / ml) was calculated based on the antigen-specific antibody amount of a standard product prepared by the following method. The results are shown in Table 2. The collected blood was centrifuged (5,000 rpm, 8 minutes), and serum was collected and used for measurement. For the nasal wash, 250 μl of physiological saline was injected into the left and right nasal cavities of each mouse (total 500 μl / mouse), collected, and then centrifuged (5,000 rpm, 8 minutes), and the supernatant was measured. Using.

<Preparation of standard for quantification of IgA antibody and IgG antibody by EIA>
After pre-breeding two BALB / c mice (Charles River Japan, female, 8 weeks old) for 1 week, influenza vaccine protein was 0.3 μg / 30 μl and cholera toxin B subunit was 0.8 μg / 30 μl. 1 week after Freund's solution was mixed continuously into the right and left nasal cavities at 15 μl / animal / dose each (total 30 μl / animal / dose), once a week for 4 weeks (4 times in total). Influenza vaccine suspended in complete adjuvant was intraperitoneally administered as an influenza vaccine protein at 0.3 μg / 300 μl / animal. Further, one week later, an influenza vaccine suspended in Freund's incomplete adjuvant was intraperitoneally administered as an influenza vaccine protein at 0.3 μg / 300 μl / mouse. One week later, both mice were blood-collected under ether anesthesia, serum was separated and mixed, and this was used as a standard for influenza vaccine protein-specific IgG antibody. In addition, intranasal washings were collected from both mice and mixed to obtain a standard product of influenza vaccine protein-specific IgA antibody. When these standards are appropriately diluted and subjected to EIA for quantification of specific IgG antibody or IgA antibody against influenza vaccine protein using the above-described influenza vaccine protein as a solid phase, the color development value (absorbance) by HRPO is 0.1. The amount of influenza vaccine protein-specific IgG antibody or IgA antibody contained in a standard product with a dilution ratio of 1 was defined as 1 unit / ml (antibody titer: U / ml), respectively. By the way, in these standards, the specific IgG antibody amount was 1 U / ml when the serum was diluted 2 million times, and the specific IgA antibody amount was 1 U / ml when the nasal wash was diluted 192 times. The detection limit of the antibody in this measurement system is 6 U / ml for IgA antibody and 21 U / ml for IgG antibody. Detection of IgA antibody and IgG antibody is also possible in the antibody titer measurement system for various antigens in the following examples. The sensitivity was a detection limit value almost the same as that in this example. In the tables showing the test results in the following Examples, when the antibody amount was below the detection limit, the antibody titer was expressed as “0 U / ml”.

As shown in Table 2, when the influenza vaccine and the TNF-α-cholesterol pullulan complex were administered nasally at the same time, production of specific IgA antibody against the influenza vaccine protein was observed in the nasal cavity, and production of specific IgG antibody Was found in the blood (Study Group 5). Influenza vaccine, TNF-α (Test group 3), TNF-α-lyotropic liquid crystal inclusion body (Test group 7), PEG-modified TNF-α (Test group 8) or cholera toxin B subunit (Test group 9) When the heel was administered nasally at the same time, production of specific IgA antibody against influenza vaccine protein was observed in the nasal cavity and production of specific IgG antibody was observed in blood. In terms of IgA antibody production, nasal administration of influenza vaccine and TNF-α-cholesterol pullulan complex was the highest, and influenza vaccine was administered without TNF-α and other adjuvants simultaneously and without adjuvant. It was significantly enhanced for any of the cases. Regarding IgG antibodies in blood, the highest was when influenza vaccine and TNF-α-cholesterol pullulan complex were administered (Study Group 5), followed by TNF-α-lyotropic liquid crystal inclusions administered simultaneously via nasal administration (Test group 7). Even when the influenza vaccine was administered without an adjuvant (Test Group 2), production of secretory IgA antibody was observed although it was weak. In the subcutaneous administration group, the production of secretory IgA antibody was observed either when the influenza vaccine was administered without an adjuvant (Test Group 10) or when administered simultaneously with the TNF-α-cholesterol pullulan complex (Test Group 11). On the other hand, the production of blood IgG antibody was enhanced in all cases, and was significantly enhanced by co-administration with TNF-α-cholesterol pullulan complex (Test Group 11). In addition, when an influenza vaccine and TNF-α-cholesterol pullulan (test group 5) or rec-TNF-α-cholesterol pullulan (test group 6) are administered simultaneously via the nasal route, specific intranasal IgA antibodies and blood There was no difference in the amount of specific IgG antibody produced. When nasal administration of influenza vaccine and TNF-α (Test Group 3) or rec-TNF-α (Test Group 4) at the same time, the amount of specific IgA antibody in the nasal cavity and specific IgG antibody production in the blood There was no difference. During the test period, the mice treated with TNF-α-lyotropic liquid crystal inclusion bodies (test group 7) were puffed and weakened, and a decrease in body weight was observed compared to the saline-treated group (test group 1). On the other hand, other influenza vaccine and / or adjuvant administration groups showed the same body weight transition as the physiological saline administration group, and no abnormal appearance was observed.

This result shows that when TNF-α is used as an adjuvant for mucosal administration, the effect of stabilizing TNF-α or DDS varies greatly depending on the method used, even if a known method for stabilizing cytokines is applied. It is shown that the most preferable adjuvant action can be obtained by the DDS formation / stabilization method for forming the complex. In addition, since the adjuvant action is stronger than the cholera toxin B subunit known to exert a remarkable action as a mucosal adjuvant, it indicates that TNF-α-cholesterol pullulan complex is extremely useful as a mucosal adjuvant. ing. Furthermore, it was found that such mucosal adjuvant activity is not different between TNF-α-cholesterol pullulan and rec-TNF-α-cholesterol pullulan. In addition, the administration of TNF-α-lyotropic liquid crystal inclusions caused weight loss and poor physical condition in mice. Therefore, there was a problem in safety when TNF-α converted to DDS by this method was used as an adjuvant for mucosal administration. It shows that there is.

<Effect of frequency of administration of TNF-α-cholesterol pullulan complex on antibody production>
In Example 2, the TNF-α-cholesterol pullulan complex (hereinafter sometimes referred to as “TNF / CHP”) among the TDS-α converted to DDS used in the test showed the strongest adjuvant action, and Since it was also excellent in terms of safety, the influence of the number of administrations on antigen-specific antibody production was examined. That is, Example 2 except that the number of administrations of the preparation in which the TNF-α-cholesterol pullulan complex and the antigen (influenza vaccine) were mixed was 1 to 4 times and nasally administered using the combinations shown in Table 3. Under the same conditions, the amount of secretory IgA antibody and blood IgG antibody in the nasal lavage fluid specific to influenza vaccine when administered to BALB / c mice were measured in the same manner as in Example 2. The results are shown in Table 3.

As shown in Table 3, when the antigen (influenza vaccine) and TNF-α-cholesterol pullulan complex were administered 1 to 4 times, depending on the number of administrations, the amount of IgA antibody in the antigen-specific intranasal lavage fluid was increased. After 2 to 4 administrations, the antigen-specific blood IgG antibody level increased depending on the number of administrations, and the amount of any antibody was the highest after 4 administrations (test groups 14 to 16). . In contrast, when an antigen and a TNF-α solution (test groups 10 to 12) or an antigen alone (test groups 6 to 8) were administered, an antigen-specific intranasal washing solution depending on the number of administrations Although the amount of IgA antibody and the amount of IgG antibody in blood increased, the amount of antibody produced was 2 to 4 times as compared with the case where TNF-α-cholesterol pullulan complex was used as an adjuvant. Was significantly lower. When physiological saline was administered, production of IgA antibody and blood IgG antibody in the antigen-specific intranasal washing solution was not observed at any number of administrations. This result shows that when a novel antigen and TNF-α-cholesterol pullulan complex are administered to a living body, in order to enhance the production of antigen-specific IgA antibody and IgG antibody, it is preferably performed twice or more, preferably three times or more. Is necessary. In addition, when administering an antigen for which immunological memory has already been established or an antigen cross-reactive with it, the TNF-α-cholesterol pullulan complex can be administered nasally at the same time to produce antibody even in a single administration. It is speculated that the production of antibodies specific to the antigen administered more efficiently can be enhanced by administration of two or more times. Further, when TNF-α-cholesterol pullulan complex is used as an adjuvant (Study Group 14), in the second administration, antigen alone (Test Group 6) or when TNF-α is used as an adjuvant (Study Group 10) Production of secretory IgA antibody in the nasal lavage fluid, which was not observed in (1), was already observed. The shortening of the period necessary for inducing production of secretory IgA antibody effective for such infection protection can impart infection protection ability to the administered individual in a short period of time, and is therefore used as a means for suppressing the spread of infectious diseases. It has been shown to be extremely useful as an adjuvant for vaccines. In addition, since no abnormalities in appearance or death were found in the mice throughout the administration period, the antigen and TNF-α-cholesterol pullulan complex were administered nasally at the same time in the dosage and usage used in this example. However, it became clear that no serious side effects occurred.

<Dose of TNF-α-cholesterol pullulan complex and antigen>
The effect of the dose of TNF-α-cholesterol pullulan complex and antigen on the production of antigen-specific IgA antibody and IgG antibody was examined. That is, using the combination shown in Table 4 for the dosage of the preparation prepared by mixing the TNF-α-cholesterol pullulan complex prepared in Example 1 and the antigen (influenza vaccine), the administration schedule once a week for 3 weeks Except for intranasal administration (3 times in total), the amount of IgA antibody and blood IgG antibody in the nasal lavage fluid specific for influenza vaccine when administered to BALB / c mice under the same conditions as in Example 2. The measurement was performed in the same manner as in Example 2. The results are shown in Table 4.

As shown in Table 4, 0.001 to 10 μg / animal / dose of antigen (influenza vaccine protein) and 0.01 to 50 μg of TNF-α-cholesterol pullulan complex (as TNF-α protein) / animal / dose simultaneously By nasal administration, production of antigen-specific IgA antibody and IgG antibody is induced, and in terms of the production amount of secretory IgA, at a dose of 0.01 to 10 μg / animal / dose, TNF- It was found that 0.1 to 50 μg of α-cholesterol pullulan complex (as TNF-α protein) / animal / dose was more effective in enhancing antibody production. In addition, when the production amount of IgG in the blood is taken together, 1 to 50 μg of TNF-α-cholesterol pullulan complex (as TNF-α protein) / mouse at a dose of 0.1 to 10 μg / animal / dose of antigen. It was found that co-administration of / times had better efficiency in enhancing antibody production. When the antigen amount was 0.1 μg or more / animal / dose, no enhancement of antibody production commensurate with the dose was observed. In addition, in the mice administered with TNF-α and antigen (Test Group 33) and the mice administered with TNF-α, cholesterol pullulan and antigen (Test Group 35), the TNF-α-cholesterol pullulan complex and the antigen Only weak antibody production was observed compared to the case of administration of. By the way, the volume of the nasal cavity of mice is usually about 30 μl, and the volume of the human nasal cavity is said to be 2 ml (dwarf) to 4 ml (adult). Therefore, considering the ratio of nasal volume, antigen and TNF-α-cholesterol When the pullulan complex is applied to a human as a vaccine, the dose per administration is about 100 times the effective dose in mice. Further, in the mice administered with TNF-α and the antigen (Test Group 33) and the mice administered with TNF-α, cholesterol pullulan and the antigen (Test Group 35), the hairs were fuzzy, weak and seemingly abnormal. In contrast to the observation, no abnormal appearance or death in mice treated with TNF-α-cholesterol pullulan complex and antigen was found throughout the administration period. It has been clarified that no serious side effects occur even when the antigen and the TNF-α-cholesterol pullulan complex are administered nasally at the same time. There is also a report that cholesterol pullulan itself has no adjuvant activity (see, for example, “Nature materials”, Vol. 9, No. 7, pages 572-578 (2010)), although it is weak in this test system. It became clear that there was an adjuvant activity.

In Examples 2 to 4, the TNF-α-cholesterol pullulan complex is extremely useful as an adjuvant for nasal administration of an antigen, and is an excellent antigen-specific blood IgG antibody and / or secretory IgA antibody. Since it was confirmed to have a production enhancing action, the effect of the antibody whose production was induced by such an adjuvant action on hemagglutination inhibition, which is a direct indicator of the ability to protect against influenza virus infection, was examined. That is, in Example 2, administration of the physiological saline simultaneously with administration of the antigen (influenza vaccine) (test group 2), TNF-α administration group (test group 3), administration of TNF-α-cholesterol pullulan complex Sera collected from the group (test group 5) and the cholera toxin B subunit administration group (test group 9) were used to measure the hemagglutination inhibition activity using a commercially available in-vitro diagnostic drug (trade name “influenza virus”). HI reagent “Seiken” (sold by Denka Seiken Co., Ltd.) was used. In accordance with the description in the attached package insert, the determination is that the hemagglutination inhibitory activity is observed when the dilution ratio of the serum is 10 times or more (“Yes”), and the inhibitory activity is not detected if it is not observed ( "No"). The results are shown in Table 5. Except for the term of hemagglutination inhibition activity in Table 5, the values in Table 2 were directly transferred.

As shown in Table 5, when the TNF-α-cholesterol pullulan complex was administered simultaneously with the administration of the antigen (influenza vaccine), type A (H1N1 and H3N2) and type B influenza contained in the vaccine used as the antigen It was determined that all viruses had inhibitory activity on hemagglutination (Test Group 5). On the other hand, when the cholera toxin B subunit was administered simultaneously with the administration of the antigen (influenza vaccine), it was determined that there was an inhibitory activity only for the hemagglutination reaction against influenza A virus (Test Group 9). When physiological saline or TNF-α was administered at the same time, it was determined that there was no inhibitory activity on hemagglutination reaction against any type of influenza virus (Test Groups 2 and 3). This result indicates that the nasal administration of the TNF-α-cholesterol pullulan complex simultaneously with the antigen can efficiently confer infection protection against all three types of influenza viruses administered as the antigen. When cholera toxin B subunit was used as an adjuvant, although the antibody titer in the blood was increased, it was insufficient for inhibiting the hemagglutination reaction against all three types of influenza viruses. Incidentally, the serum obtained in Example 4 was similarly checked for the presence or absence of hemagglutination inhibition activity. When the IgG antibody amount exceeded 20,000 U / ml, all of the three types of influenza viruses were tested. Inhibitory activity of hemagglutination was observed.

<Effects of nasal administration of influenza vaccine and TNF-α-cholesterol pullulan complex on influenza virus infection 1>
In Examples 2 to 5, the TNF-α-cholesterol pullulan complex is extremely useful as an adjuvant for nasal administration of an antigen, and is useful for preventing infection and preventing onset of disease and its seriousness. In addition, since it was confirmed that the production of the antigen-specific blood IgG antibody and / or secretory IgA antibody is excellent, the adjuvant action of the TNF-α-cholesterol pullulan complex is actually in vivo. It was confirmed by using a mouse infection model that is widely used as a model animal for human influenza virus infection that it is useful for induction of the protective ability against infection. In other words, 45 BALB / c mice (Nippon Charles River Co., Ltd., female, 8 weeks old) were randomly divided into 9 groups of 5 each, and the influenza vaccine solution and TNF-α-cholesterol pullulan complex were simultaneously administered. The administration schedule was 1 to 2 times per week (2 to 4 times in test groups 7 to 9). As a control 1, in place of the influenza vaccine solution and the TNF-α-cholesterol pullulan complex, physiological saline was simultaneously administered once a week, 2 to 4 times (test groups 1 to 3). As a control 2, an influenza vaccine solution and physiological saline instead of the TNF-α-cholesterol pullulan complex were simultaneously administered once a week, 2 to 4 times (test groups 4 to 6). The doses of TNF-α-cholesterol pullulan complex, influenza vaccine and physiological saline were administered nasally at 30 μl / animal / dose.

Influenza vaccine solutions used as antigens are commercially available influenza vaccines (trade name “influenza HA vaccine”, sold by Denka Seiken Co., Ltd., manufactured as A / Brisbane / 59/2007 (H1N1 type) (A Soviet type), A / Uruguay / 716/2007 (H3N2 type) strain (formulation using A Hong Kong type) and B / Brisbane / 60/2008 strain) as a virus-derived protein of each strain to be 0.3 μg / 30 μl Dilute with saline and administer.

As the TNF-α-cholesterol pullulan complex used as an adjuvant, the TNF-α protein prepared by the method of Example 1 was diluted with physiological saline to be 5 μg / 30 μl and administered. After 1 week of preliminary breeding, administration is started from mice set to 4 doses, administration start time is delayed by 1 week, and administration is also started to mice set to 3 and 2 doses. Was the same in all test groups.

One week after the end of the prescribed number of administrations, influenza virus (PR8 strain (H1N1 type), 160 pfu / 50 μl (corresponding to 10 times the LD ₅₀ amount for 6 to 7-week-old mice) was administered nasally, and then Confirmation of survival and body weight measurement were carried out daily, relative values were determined with the number of mice at the time of virus administration being 100%, and the average value of each group is shown in Table 6. The body weight at the time of virus administration is also shown in Table 6. The relative value of the body weight when it is 100% is obtained, subtracted from 100%, and the average value of each group is shown as the rate of change in body weight in Table 7. In this example, the value of the body weight change rate in Table 7 is negative. It means that the lower the rate of change, the smaller the effect on the body weight, the less the influence on the body weight, and the milder the symptoms were even when the virus infection was stopped or infected. 25% compared to before virus infection (rate of change: -25%) when decreased from, mice considered to have died were euthanized.

As shown in Tables 6 and 7, when the influenza vaccine was administered intranasally 3 or 4 times per week on an administration schedule once a week, and the TNF-α-cholesterol pullulan complex was administered intranasally as an adjuvant, There were no dead animals in the mice, and the rate of change in body weight was extremely low at about -5% (Test Groups 8 and 9). When the influenza vaccine was administered nasally twice at the same time as the once-weekly administration schedule, and the TNF-α-cholesterol pullulan complex was administered nasally, mice did not die, and 6 and 7 after virus administration Although a decrease in body weight of up to 12% (change rate: -12%) was observed on the day, it started to increase after the 8th day (Study Group 7). On the other hand, when the influenza vaccine and physiological saline were administered nasally at the same time two times (test group 4) or three times (test group 5), a dead individual occurred 8 to 9 days after the virus was administered, 60% and 80% of the individuals died, respectively. In this case, the body weight of the individuals that survived even after the 10th day of virus administration decreased by 20% or more (change rate: -20%) on the 7th and 8th days after the virus administration, and then started to increase. When only the influenza vaccine was administered four times (Test Group 6) and when only the physiological saline was administered (Test Groups 1 to 3), all individuals died within 7 to 9 days after the virus was administered. . This result shows that nasal administration of the TNF-α-cholesterol pullulan complex simultaneously with the influenza vaccine dramatically enhances the ability to protect against influenza virus compared to the single administration of the vaccine. Considering the degree of change in body weight, the dose used in this example is more likely to be administered 3 or 4 times than the influenza vaccine and TNF-α-cholesterol pullulan complex twice. It shows that the ability to protect against infection can be effectively induced in the living body.

<Influence 2 on influenza virus infection by nasal administration of influenza vaccine and TNF-α-cholesterol pullulan complex>
In Example 6, it was clarified that the influenza vaccine and TNF-α-cholesterol pullulan complex can be effectively administered at the same time by nasal administration, so that the ability to protect against influenza virus can be effectively enhanced. A test was conducted in which the usefulness of the TNF-α-cholesterol pullulan complex as an adjuvant for mucosal administration was compared with that of cholera toxin B subunit, which has been confirmed to be excellent in action and effect as an adjuvant for mucosal administration. That is, 50 BALB / c mice (Charles River Japan Inc., female, 8 weeks old) were randomly divided into 5 groups of 10 mice and administered nasally with combinations of antigens and adjuvants shown in Tables 8 and 9 And the effects on influenza virus infection were evaluated. In the test, after pre-breeding the mice for 1 week, adjuvant and antigen were administered 3 times in a once weekly administration schedule, influenza virus was administered 1 week after the 3rd administration, and the observation period after virus administration was 15 The same method as in Example 6 was performed except that the period was one day. The survival rate is shown in Table 8, and the weight change rate is shown in Table 9. The cholera toxin B subunit used as a positive control (Wako Pure Chemical Industries, Ltd., lot number CDH6462) was diluted with physiological saline to a protein concentration of 0.8 μg / 30 μl, and then 0.8 μg / 30 μl / The animals / nasal administration was performed.

As shown in Tables 8 and 9, mice survived when the influenza vaccine was administered nasally three times on a once weekly dosing schedule and simultaneously with nasal administration of TNF-α-cholesterol pullulan complex as an adjuvant. The rate was extremely high at 90% and the rate of change in body weight was also extremely low at about -7% (Test Group 4). When the nasal administration of the influenza vaccine and nasal administration of cholera toxin B subunit instead of the TNF-α-cholesterol pullulan complex, the survival rate of the mice was as high as 90%, and the rate of change in body weight was also − It is extremely low, about 5% (Study Group 5), and there was no significant difference in survival rate or change in body weight between the two groups. TNF-α-cholesterol pullulan complex was administered with cholera toxin B subunit. The strong adjuvant activity similar to the case was confirmed. When only physiological saline was administered three times (Test Group 1), when TNF-α-cholesterol pullulan complex and physiological saline were administered nasally three times simultaneously (Test Group 2), and influenza vaccine and physiological In the case of nasally administering the saline solution three times at the same time (Test Group 3), all individuals died by 10 days after the influenza virus administration. This result shows that nasal administration of TNF-α cholesterol pullulan complex at the same time as the influenza vaccine induces more effective protection against influenza virus than administration of the vaccine alone. Furthermore, the influenza virus PR8 strain (H1N1 type) used for the infection and the A / Brisbane / 59/2007 (H1N1 type) (A Soviet type) virus contained in the influenza vaccine administered as an antigen differ in antigenicity. Regardless (see, for example, “Journal of Virology”, Mar., pages 2910-2919 (2005)), in the serum of mice immunized with an influenza vaccine that does not contain a protein derived from the influenza virus PR8 strain, Since the induction of an IgG antibody that inhibits aggregation was confirmed, the TNF-α-cholesterol pullulan complex is useful as a mucosal adjuvant that induces cross-protection ability, and causes an antigenic mutation that causes a pandemic. Sub-type I To be useful in the induction of infection defenses against influenza virus it revealed. In addition, the adjuvant action of TNF-α-cholesterol pullulan complex is equivalent to that of cholera toxin B subunit, indicating that it is extremely useful as a nasal adjuvant. Furthermore, these results are in good agreement with the results relating to the enhancement of antigen-specific blood IgG antibody and / or secretory IgA antibody production in Example 2 and the results of hemagglutination inhibition activity in Example 5. From the above, the enhancement of infection-protecting ability against pathogenic microorganisms enhanced by the adjuvant action of TNF-α-cholesterol pullulan complex is achieved by antigen-specific blood IgG antibody and / or secretory IgA antibody against antigens derived from such microorganisms It was judged to be due to the enhancement of production.

<Effect of TNF-α-cholesterol pullulan complex administration on antibody production against antigens derived from various pathogenic microorganisms 1>
From the results of Examples 2 to 7, nasal administration of the TNF-α-cholesterol pullulan complex to the living body enhanced the production of antigen-specific secretory IgA antibody against the nasally administered antigen and IgG antibody in the serum, As a result, it has been clarified that a strong infection-protective ability can be efficiently induced against pathogenic microorganisms having the administered antigen. Thus, antigens derived from pathogenic microorganisms other than influenza administered with TNF-α-cholesterol pullulan complex The effect on antibody production at the time of administration was examined. Using the hepatitis A vaccine and diphtheria toxoid shown below, the adjuvant effect by nasal administration of TNF-α-cholesterol pullulan complex was compared with the adjuvant effect when cholera toxin B subunit was used. The antigen used for the test and the preparation method thereof are shown below.

<Hepatitis A vaccine>
Commercially available dry tissue culture inactivated hepatitis A vaccine (trade name “Aimgen”, manufactured by Chemical and Serum Therapy Laboratories, may be abbreviated as “HAV”). Diluted by adding 0.7 ml. HAV was stored at 4 ° C. after preparation. Immediately before administration, 80 μl of TNF / CHP (250 μg / ml as TNF-α protein) or CTB (40 μg / ml) was mixed with 40 μl of HAV (1 μg / ml as HAV-derived protein) and administered at 30 μl / animal ( As HAV, 0.01 μg / animal / time). Nasal administration was performed continuously for 4 weeks (total 4 times) on a once-weekly administration schedule. Hereinafter, the administration frequency, administration method, and administration schedule were the same for all antigens.

<Diphtheria toxoid>
0.5 mg of a lyophilized product of a commercially available diphtheria toxoid (sold by LIST BIOLOGICAL LABORATORIES INC., Hereinafter sometimes abbreviated as “DT”) was diluted to 0.1 mg / ml with sterilized water and stored at 4 ° C. Immediately before administration, 40 μl of DT solution (0.1 mg / ml as diphtheria toxoid protein) was mixed with 80 μl of TNF / CHP (250 μg / ml as TNF-α protein) or 80 μl of CTB (40 μg / ml), and 30 μl / animal (1 μg / animal / dose as diphtheria toxoid protein).

<Test method>
72 BALB / c mice (Nippon Charles River Co., Ltd., 8 weeks old, female) were randomly divided into 9 groups of 8 mice. After one week of preliminary breeding, a test sample containing any one of the two antigens prepared above and either TNF / CHP as an adjuvant was placed in each of the right and left holes of the nose at 15 μl / animal / dose (total). 30 μl / animal / dose), administered once a week for 4 weeks (4 times in total). In addition, physiological saline was used as an adjuvant control. One week after the fourth administration of the test sample, the mouse was dissected under ether anesthesia, blood and nasal washes were collected, and the antigen-specific IgA antibody and IgG antibody amounts were administered to the mouse in the solid phase. It measured by the enzyme antibody method. The amount of the antibody was separately mixed for each antigen with the antigen used for the test in BALB / c mice and cholera toxin B subunit, and each left and right nasal cavity was 15 μl / unit (total 30 μl / unit / unit), Administer once a week for 4 weeks (4 times in total). Similarly, blood and nasal lavage fluid are collected. In the case of using antigen-specific IgA antibody and hepatitis A vaccine as the antigen, blood The amount of IgM antibody, and the amount of IgG antibody in blood when diphtheria toxoid was used, were measured by an enzyme antibody method using an antigen administered to mice as a solid phase. The results are also shown in Table 10. In hepatitis A, the production of IgM antibody in blood is usually used as an indicator of infection (for example, “Journal of the Japan Society for Clinical Laboratory Automation”, Vol. 16, No. 3, pp. 194-200 (1991). Also in this example, the amount of antigen-specific blood IgM antibody produced was measured to determine the adjuvant effect. The detection limit of the hepatitis A vaccine-specific IgM antibody in this example was 21 U / ml, which was comparable to the sensitivity of the antigen-specific IgG antibody detection method used in other examples.

As shown in Table 10, when either the hepatitis A vaccine or diphtheria toxoid as an antigen and TNF-α-cholesterol pullulan complex as an adjuvant are administered nasally, specific secretion for each administered antigen The amount of type IgA antibody and blood IgM antibody or IgG antibody increased. On the other hand, when cholera toxin B subunit or TNF-α is administered nasally as an adjuvant together with these antigens, the amount of secretory IgA antibody and the amount of IgM antibody or IgG antibody in the blood specific to each administered antigen However, the increase was significantly lower than when TNF-α-cholesterol pullulan complex was administered as an adjuvant. When physiological saline was administered together with the antigen in place of the adjuvant, an increase in the amount of secretory IgA antibody and blood IgM antibody or IgG antibody specific to each administered antigen was not observed. In addition, since no abnormal appearance or death was found in the mice throughout the administration period, these antigens used in the test and the TNF-α-cholesterol pullulan complex were used in the dosage and usage used in this Example. At the same time, it became clear that no serious side effects occurred even when administered intranasally.

<Effect of TNF-α-cholesterol pullulan complex administration on antibody production against antigens derived from various pathogenic microorganisms 2>
Simultaneously, these antigens were administered intranasally in the same manner as in Example 8, except that various commercially available vaccines shown in Table 11 were used as antigens, and TNF-α-cholesterol pullulan complex or TNF-α was used as an adjuvant. Then, the effects of nasal administration of TNF-α-cholesterol pullulan complex as an adjuvant on the production of antigen-specific secretory IgA antibody and blood IgG antibody against the administered antigen were examined. The following antigens were used for the test.

<Antigen>
Pneumococcal vaccine (trade name “Pneumobax NP”, manufactured by MSD Co., Ltd.), dry BCG vaccine (trade name “dry BCG vaccine”, manufactured by Nippon BCG Co., Ltd.), dry rabies vaccine (trade name “inactivated dry tissue culture” Rabies vaccine ”, manufactured by Astellas Pharma Inc.), dry attenuated varicella vaccine (trade name“ Dry attenuated varicella vaccine “Biken” ”, Osaka University Microbial Disease Research Association) and Japanese encephalitis vaccine (trade name“ Jebic ”) V ”(produced by the Osaka University Microbial Diseases Research Association) were diluted with physiological saline so that the concentrations shown in Table 11 were used. The results are also shown in Table 11.

As shown in Table 11, pneumococcal vaccine (Test group 3), dry BCG vaccine (Test group 6), dry rabies vaccine (Test group 9), dry attenuated varicella vaccine (Test group 12) and Japanese encephalitis vaccine (Test) When the TNF-α-cholesterol pullulan complex was administered as an adjuvant together with any of the groups 15), the amount of secretory IgA antibody and the amount of blood IgG antibody specific to each administered antigen increased. In contrast, when TNF-α was used as an adjuvant, only a slight increase in the amount of secretory IgA antibody and blood IgG antibody specific to the antigen was observed in any antigen. . An antigen-specific antibody titer was not increased when physiological saline was administered instead of the adjuvant. In addition, since no abnormal appearance or death was found in the mice throughout the administration period, these antigens used in the test and the TNF-α-cholesterol pullulan complex were used in the dosage and usage used in this Example. At the same time, it became clear that no serious side effects occurred even when administered intranasally.

<Effect of administration of TNF-α-cholesterol pullulan complex on antibody production by nasal administration of antigen (allergen)>
In Examples 8 and 9, administration of TNF-α-cholesterol pullulan complex can effectively enhance the production of secretory IgA antibody, blood IgG antibody or IgM antibody by nasal administration of various vaccines, Since it was clarified that the enhancement effect was superior to that of TNF-α, in this example, the effect on the antibody production when allergen was administered as an antigen was determined using cholera toxin B subunit as an adjuvant. Compared to the case. As the allergen, a representative cedar pollen-derived allergen (hereinafter referred to as “SBP”) was used.
<Test method>
4 hours after nasal administration of TNF-α-cholesterol pullulan complex or cholera toxin B subunit as an adjuvant, using allergens derived from cedar pollen in the doses shown in Table 12 instead of antigens derived from pathogenic microorganisms Thereafter, a sensitization test was carried out by the same method as in Example 9 except that cedar pollen-derived allergen was administered as an antigen nasally, and the amount of secretory IgA antibody and secretory type in the administered antigen-specific intranasal washing solution The amount of IgG antibody, the amount of IgA antibody in blood, and the amount of IgG antibody in blood were measured. In addition, the standard for antigen-specific secretory IgA antibody and blood IgG antibody was prepared by the same method as the method for preparing the standard product of IgA antibody and IgG antibody. The results are shown in Table 12.

As shown in Table 12, when TNF-α-cholesterol pullulan complex was administered nasally simultaneously with administration of the antigen (cedar pollen-derived allergen: SBP) (test group 2), antigen-specific blood Production of IgG and IgA antibodies as well as IgA antibodies in intranasal lavage fluid was enhanced. Also when cholera toxin B subunit (CTB) was administered nasally (Test Group 4), production of IgG antibody and IgA antibody in blood and IgA antibody in nasal lavage fluid was enhanced. The amount of secretory IgA antibody and blood IgA antibody produced was not significantly different between administration of TNF-α-cholesterol pullulan complex and CTB as an adjuvant, but antigen-specific blood The amount of medium IgG antibody was much higher when the TNF-α-cholesterol pullulan complex was administered. Such a transition in antibody titer indicates that TNF-α-cholesterol pullulan complex is also useful as an adjuvant when using an allergen as an antigen. Furthermore, the test results of these Examples 1 to 10 show that TNF-α-cholesterol pullulan complex is a versatile mucosal administration adjuvant for enhancing antibody production against vaccines, toxic components, allergens and the like. It is useful. In addition, since no abnormalities in appearance or death were found in the mice throughout the administration period, the antigen and TNF-α-cholesterol pullulan complex were administered nasally at the same time in the dosage and usage used in this example. However, it became clear that no serious side effects occurred.

<Acute toxicity test>
A test was conducted to confirm the safety of the adjuvant for mucosal administration according to the present invention containing TNF-α-cholesterol pullulan complex as an active ingredient. That is, BALB / c mice (Nippon Charles River Co., Ltd., 8 weeks old, male and female, average body weight 20 g) each 35 were randomly divided into 7 groups of 5 each, and 5 males and 2 females each. The same TNF-α-cholesterol pullulan complex as used in Example 1 was diluted with physiological saline and administered orally or subcutaneously at 200 μl / animal (Test Groups 5 and 6). Five males and two females each were orally or subcutaneously administered with 200 μl / mouse of saline containing no adjuvant (Test Groups 2 and 3). The same TNF-α-cholesterol pullulan complex as used in Example 1 was diluted in physiological saline and administered to both nasal cavities in a volume of 15 μl for each group of 5 mice per group (total 30 μl). (Study group 7). In each group of 5 males and 5 males, 15 μl of saline containing no adjuvant was administered intranasally (total 30 μl / mouse) (Test group 4). The remaining 5 males and 1 female were left untreated as a control (Test Group 1). After observing the appearance of these mice for 48 hours after administration of physiological saline with or without TNF-α-cholesterol pullulan complex, the mice were necropsied by a conventional method, and administration sites of antigen and adjuvant, and pathological tissues of major organs In addition to confirming the presence or absence of abnormalities by observing the blood, blood and urine were collected, and clinical tests were performed as indicators of bone marrow function, kidney function, and liver function. In addition, the untreated mice were similarly observed for appearance and histopathology, and were subjected to clinical examination as an index of bone marrow function, kidney function and liver function. Table 13 also shows the presence or absence of abnormalities when compared with untreated mice. Since the test results showed no difference between males and females, the results are summarized in Table 13 when administered to males and females.

As is apparent from Table 13, even when the TNF-α-cholesterol pullulan complex was administered orally or nasally, no change in appearance was observed from immediately after administration until the end of observation, and the site of administration and major organs were not observed. There were no histopathological abnormalities, and there were no differences in the laboratory test values as indices of bone marrow function, kidney function and liver function from the control mice (Test Group 1). No toxicologically significant findings were found. This result shows that the TNF-α-cholesterol pullulan complex is extremely safe even when administered orally and nasally to humans. In addition, when TNF-α-cholesterol pullulan complex was administered subcutaneously (Study Group 6), although it was not determined that the systemic abnormality was toxicologically, neutrophils were observed to be mild by histopathological observation. Since infiltration was observed, it is presumed that slight inflammation is induced at the administration site at this dose. An acute toxicity test was conducted under the same conditions as in Example 11 except that rec-TNF-α was used. As a result, no significant difference was observed from that in Example 11 using the wild type. It was.

<Adjuvant formulation: solution form>
Weigh an appropriate amount of cholesterol pullulan (trade name “PUREBRIGHT CP-100T”, sold by NOF Corporation) and suspend it in Dulbecco's phosphate buffered saline (D-PBS (−)) to 20 mg / ml. After dissolution in a warm water bath at 55 ° C. for 16 hours, the solution was sterilized by filtration using a membrane filter having a pore size of 0.22 μm. Either 7 ml of this cholesterol pullulan solution and 2 ml of TNF-α used in Example 1 or rec-TNF-α used in Example 2 (both TNF-α protein amount: 1.454 mg / ml) dissolved in D-PBS. The heels were mixed, sterilized by filtration using a membrane filter having a pore size of 0.22 μm, and then reacted at 37 ° C. for 5 days to prepare two TNF-α-cholesterol pullulan complex-containing solutions. Each of these solutions is diluted with physiological saline for injection (trade name “Otsuka Seikatsu”, sold by Otsuka Pharmaceutical Co., Ltd.) to obtain 2.5, 25 and 250 μg / ml solutions as TNF-α protein. An adjuvant for mucosal administration was prepared by filling with 0.5 ml / vial.

This product is used for mucosal administration of these antigens for the purpose of enhancing antibody production against antigens such as vaccines, toxic components, and allergens used to prevent infection of pathogenic microorganisms in vertebrates, particularly humans. Usually, 0.01 ml to 0.2 ml per adult is administered to the mucosa simultaneously with or before or after the antigen, whereby the antigen-specific blood IgG antibody and / or IgM antibody administered, and Since the production of secretory IgA antibody in the mucosal tissue can be effectively enhanced, it is extremely useful for acquiring infection protection ability (including cross-protection ability) against pathogenic microorganisms in vertebrates, especially humans, and for allergic diseases. Useful. In addition, this product is a highly safe adjuvant preparation that does not cause serious side effects even when administered to a living body together with various antigens.

<Adjuvant formulation: lyophilized form>
In the same manner as in Example 12, TNF-α-cholesterol pullulan complex and rec-TNF-α-cholesterol pullulan complex were made into TNF-α protein solutions of 1, 10 and 100 μg / ml, respectively. It was filled with 0.5 ml / vial and freeze-dried by a conventional method to prepare an adjuvant for mucosal administration.

This product dissolves in purified water for injection at the time of use, and aims to enhance the production of antibodies against antigens such as vaccines, toxic components and allergens used to prevent infection of pathogenic microorganisms in vertebrates, especially humans When these antigens are administered to the mucosa, 0.01 ml to 0.2 ml of the antigen is administered to the mucosa simultaneously with or before or after the antigen, whereby the antigen-specific blood IgG antibody and / or IgM administered Since it is possible to effectively enhance the production of antibodies and secretory IgA antibodies in mucosal tissues, it is possible to acquire the ability to prevent infection (including cross-protection ability) against pathogenic microorganisms in vertebrates, particularly humans, It is extremely useful for diseases. In addition, this product is a highly safe adjuvant preparation that does not cause serious side effects even when administered to a living body together with various antigens.

<Vaccine preparation for mucosal administration: solution form>
In the same manner as in Example 12, the TNF-α-cholesterol pullulan complex and the rec-TNF-α-cholesterol pullulan complex were each made into solutions of 2, 20, and 200 μg / ml as TNF-α protein, A solution obtained by diluting each of the components shown in Table 14 below as an antigen with physiological saline and mixing an equal amount of any one of vaccines, toxoid, amyloid β peptide or allergen diluted to the protein concentration shown in Table 14 Each was filled with 0.5 ml / vial to prepare a composition for enhancing antibody production for mucosal administration.

When this product was administered nasally at 15 μl / dose / mouse 2 to 4 times in the right and left nasal cavities of the mouse according to the method of Example 2, each composition contained the antigen contained therein. Production of specific, secretory IgA antibody, blood IgG antibody and / or blood IgM antibody was enhanced, and the production amount of these antibodies was enhanced depending on the number of administrations. This product is usually antigen-specific to the combined antigen by mucosal administration of 0.01 ml to 0.2 ml per adult or adult, about 1 to 4 times, preferably 3 to 4 times. Can effectively enhance the production of blood IgG antibody and IgA antibody, and secretory IgA antibody in mucosal tissue, so that when the administered antigen is a vaccine, infection with a pathogenic microorganism from which the antigen is derived It is extremely useful for obtaining protective ability (including cross-protective ability), and for preventing the onset of infection or suppressing its seriousness. Moreover, when the administered antigen is a toxoid, the onset of the infection by the toxin from which the antigen originates can be prevented effectively. When the administered antigen is amyloid β peptide, it is useful for preventing the onset of various diseases caused by the toxicity of amyloid β peptide such as Alzheimer's disease or suppressing the seriousness. When the administered antigen is an allergen, it is useful for suppressing the onset of allergies caused by such allergens or reducing the symptoms. This product is a highly safe composition for mucosal administration that does not cause serious side effects even when administered to a living body.

<Vaccine preparation for mucosal administration: lyophilized product form>
In the same manner as in Example 12, the TNF-α-cholesterol pullulan complex and the rec-TNF-α-cholesterol pullulan complex were each made into solutions of 2.5, 25, and 250 μg / ml as TNF-α protein, respectively. In addition, any of the vaccines, toxoid, amyloid β peptide, or allergen that are diluted with physiological saline and diluted to the protein concentration shown in Table 14 except for the oral live polio vaccine shown in Table 14 above as antigens. Each solution mixed with an equal amount of seeds was filled in 0.5 ml / vial, and this was freeze-dried by a conventional method to prepare a vaccine preparation for mucosal administration.

This product was dissolved in 0.5 ml of purified water for injection and administered nasally 2 to 4 times at 15 μl / dose / mouse into the right and left nasal cavities of mice according to the method of Example 2. In any composition, the production of antigen-specific, secretory IgA antibody, blood IgG antibody and / or blood IgM antibody contained in the composition is enhanced, and the production amount of these antibodies depends on the number of administrations. Enhanced. This product dissolves in purified water for injection at the time of use. Usually, 0.01 ml to 0.2 ml of adult or adult animal is administered to mucous membranes 1 to 4 times, preferably 3 to 4 times. By doing so, it is possible to effectively enhance the production of antigen-specific blood IgG antibody and IgA antibody against the formulated antigen, and secretory IgA antibody in mucosal tissue. In addition, this product can effectively enhance the production of blood IgG antibodies specific to the administered antigen by administering to the site other than the mucosal tissue by a transdermal route. In the case of vaccines for infection protection against microorganisms, it is extremely useful for acquiring infection protection ability (including cross-protection ability) against pathogenic microorganisms from which the antigen is derived, as well as preventing the onset of infections and suppressing their severity. It is. Moreover, when the administered antigen is a toxoid, the onset of the disease by the toxin from which the antigen is derived can be effectively prevented. When the administered antigen is amyloid β peptide, it is useful for preventing the onset of various diseases caused by cytotoxicity of amyloid β peptide such as Alzheimer's disease or for suppressing the seriousness. When the administered antigen is an allergen, it is useful for suppressing the onset of allergies caused by such allergens or reducing the symptoms. This product is a highly safe composition for mucosal administration that does not cause serious side effects even when administered to a living body.

As described above, the transmucosal adjuvant of the present invention containing TNF-α-cholesterol pullulan complex as an active ingredient effectively enhances antibody production against an antigen administered to the mucosa or to other sites by a transdermal route. A composition for enhancing antibody production comprising an adjuvant that can be used as an active ingredient and such an adjuvant and an antigen as an active ingredient is a composition that can effectively enhance antibody production against an antigen administered to a mucosa or other site by a transdermal route. As a product, it can be used in the industry for producing pharmaceuticals such as vaccines and in the industry for producing antibodies for reagents and clinical diagnostics. The present invention is an invention that exhibits such remarkable effects, and is a truly significant invention that contributes greatly to the world.

Claims

A composition for enhancing antibody production, which comprises an antigen and a complex comprising Tsumor Necrosis Factor-α (TNF-α) and cholesterol pullulan.
The composition for enhancing antibody production according to claim 1, wherein the antibody is a secretory immunoglobulin A antibody.
The composition for enhancing antibody production according to claim 1 or 2, wherein the cholesterol pullulan has one or more and two or less cholesteryl groups introduced per 100 molecules of glucose in the pullulan molecule.
Furthermore, the composition for antibody production enhancement in any one of Claim 1 thru | or 3 formed by mix | blending 1 or 2 or more types of components accept | permitted pharmaceutically.
The composition for enhancing antibody production according to any one of claims 1 to 4, wherein the antigen is derived from a pathogenic microorganism.
6. The composition for enhancing antibody production according to claim 5, wherein the antigen derived from the pathogenic microorganism has an ability of inducing antibody production effective in protecting against the pathogenic microorganism of the pathogenic microorganism and / or its subtype. .
Antigens capable of inducing antibody production effective in protecting against pathogenic microorganisms and / or subtypes of pathogenic microorganisms are antigens used as vaccines for protecting the infection of those microorganisms or production of those microorganisms The composition for enhancing antibody production according to claim 6, wherein the composition is any one or more selected from toxoids for enhancing production of neutralizing antibody of toxin.
The composition for enhancing antibody production according to any one of claims 1 to 7, which is for mucosal administration.
The composition for enhancing antibody production according to claim 8, which is a nasal administration or oral administration form.
An adjuvant containing a complex composed of Tsumore Necrosis Factor-α (TNF-α) and cholesterol pullulan.
The adjuvant according to claim 10, wherein the cholesterol pullulan has one or more and two or less cholesteryl groups introduced per 100 molecules of glucose in the pullulan molecule.
Furthermore, the adjuvant of Claim 10 or 11 formed by mix | blending 1 or 2 or more types of components accept | permitted pharmaceutically.