JP2008127277A - Immunostimulation promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an immunostimulation promoter. <P>SOLUTION: The immunostimulation promoter comprises a low-molecular-weight monocarboxylic acid compound such as L-lactic acid, etc., as an active ingredient, has an immunostimulatory action by a TLR2 agonist and/or TLR4 agonist, in detail, enhances an IL-23 and an IL-17 secretion promotion actions and is useful as a therapeutic agent or prophylactic for microbial infectious diseases such as opportunistic infectious diseases. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an immunostimulation accelerator.

As immunotherapy for cancer, treatment using effector cells such as LAK (Lymphokine-activated killer cells) therapy has been actively performed, but a Toll-like receptor (Toll-) that has a bacterial or viral component as an agonist. Like Receptor (TLR), cancer immunotherapy targeting antigen-presenting cells such as dendritic cells and monocytes / macrophages has attracted attention. That is, TLR uses bacteria-derived components having an immunostimulatory effect as agonists, TLR agonists as non-specific innate immune activation therapy agents, as non-specific cancer immunotherapy not limited to cancer types, or cancer It can be used as an adjuvant used in combination with an antigenic peptide (cancer vaccine).
Specifically, bacterial lipopeptides, peptidoglycans, or cell wall skeleton components (CWS) are listed as Toll-like receptor 2 (TLR2) agonists, and lipopolysaccharide (LPS) is listed as a Toll-like receptor 4 (TLR4) agonist. (See Non-Patent Document 1). Among them, Pisibanil (registered trademark) (OK-432), Bacillus calmette-guerin cell wall skeleton (BCG-CWS), and the like are used clinically.

However, there are differences in the immunostimulatory reaction by a TLR agonist depending on the type of cancer and the person. For example, when BCG-CWS is administered intradermally, it is known that the appearance of a skin reaction (erosion) occurring at the administration site or an increase reaction of blood interferon γ is often delayed in lung cancer patients. Therefore, there is a need for a therapeutic method that enhances the immunostimulatory action of a TLR agonist.
In addition, TLR2 agonists or TLR4 agonists act on monocytes, macrophages, dendritic cells, etc., enhance antigen presentation ability, increase expression of costimulatory molecules such as CD80 and CD86, induce secretion of various cytokines, and T cells It is known that immunostimulation is achieved by differentiating activation of. Matsumoto et al. Have reported that when peripheral blood of lung cancer patients is stimulated with BCG-CWS, excessive secretion of IL-12 / 23p40 subunit is observed (see Non-Patent Document 2).
On the other hand, L-lactic acid is involved in collagen production and angiogenesis during wound healing (see Non-Patent Document 3), reducing the expression of CD1 molecules on the surface of dendritic cells, It has been reported that IL-12 production is suppressed when LPS stimulation is applied to dendritic cells (see Non-Patent Document 4). In addition, sodium lactate acts on U937 histiocytes, and LPS-induced matrix metalloproteinase-1 (matrix metalloproteinase-1), IL-6 and IL-1 secretion are enhanced, and the activity is It has been reported that L-lactic acid cannot be seen (see Non-Patent Document 5).
However, it has not been known at all whether L-lactic acid enhances the immunostimulatory activity of a TLR2 agonist or a TLR4 agonist.
S. Tsuji et al., Infect Immun. Vol. 68 No.12 p6883-90, 2000 M. Matsumoto et al., Int. Immunopharmacol. Vol.1 No.8 p1559-69, 2001 Odilo Trabold et al., Wound Repair and Regeneration vol.11, No.6, p504-509, 2003 Eva Gottfried et al., Blood Vol. 107, No. 5, 2013-2021, 2006 Alena Nereika et al., Am. J. Physiol. Endocrinol. Metab. Vol. 289, E534-542, 2005

  An object of the present invention is to provide an agent that enhances the immunostimulatory action of an immunostimulation promoter, specifically, a TLR2 agonist and / or a TLR4 agonist.

The present inventors considered that the difference in the reactivity of cancer patients to BCG-CWS may be due to the physiological action of substances contained in cancer cells, and derived from cancer cells in peripheral blood mononuclear cells (PBMC). The culture supernatant was added and the responsiveness to BCG-CWS stimulation was examined. As a result, it was found that a low molecular weight fraction of the culture supernatant of a cell line established from lung cancer patients: CADO-LC10 cells enhances the expression of IL-23p19 at the transcription level (mRNA amount). As a result of further intensive studies, it is clear that the substance having an IL-23p19 expression enhancing activity secreted from the culture supernatant of lung cancer is a low molecular weight monocarboxylic acid compound such as L-lactic acid. Became. In other words, L-lactic acid in the presence of BCG-CWS increased IL-23 secretion and IL-17 secretion from T cells. This result shows that the effect of BCG-CWS is enhanced by the action of L-lactic acid. Therefore, it was found that a combination of a low molecular weight monocarboxylic acid compound such as L-lactic acid and BCG-CWS can be an effective immunostimulatory therapy.
The present invention has been completed based on the above findings.

That is, the present invention
[1] An immunostimulation accelerator containing a low molecular weight monocarboxylic acid compound as an active ingredient;
[2] The immunostimulation accelerator according to claim 1, wherein the low molecular weight monocarboxylic acid compound is L-lactic acid, acetic acid or thioglycolic acid;
[3] The immunostimulation promoter according to [1] or [2], which is an IL-23 and / or IL-17 secretion promoter;
[4] The immunostimulation promoter according to any one of [1] to [3], wherein the immunostimulation is by a TLR2 agonist and / or a TLR4 agonist;
[5] The immunostimulation promoter according to [4], wherein the TLR2 agonist is bacterial lipopeptide, peptidoglycan or BCG-CWS, and the TLR4 agonist is LPS;
[6] The immunostimulation promoter according to any one of [1] to [3], wherein the immunostimulation is caused by a cancer antigen;
[7] The immunostimulation promoter according to any one of [1] to [3], wherein the immunostimulation is by a microorganism or a microbial vaccine;
[8] The immunostimulation promoter according to any one of [1] to [6], which is a cancer immunotherapy agent;
[9] The immunostimulation promoter according to any one of [1] to [5] and [7], which is a therapeutic agent or preventive agent for microbial infections;
[10] The immunostimulation promoter according to [9], wherein the microbial infection is an opportunistic infection;
[11] The immunostimulatory promoter according to [10], wherein the opportunistic infection is Klebsiella pneumonia, cryptococcus infection, tuberculosis, toxoplasma infection, or Candida infection;
[12] A TLR2 agonist and / or TLR4 agonist activity enhancer comprising a low molecular weight monocarboxylic acid as an active ingredient;
[13] The TLR2 agonist and / or TLR4 agonist activity enhancer according to [12], wherein the TLR2 agonist is bacterial lipopeptide, peptidoglycan or BCG-CWS, and the TLR4 agonist is LPS;
[14] The TLR2 agonist and / or TLR4 agonist activity enhancer according to [12] or [13], wherein the low molecular weight monocarboxylic acid compound is L-lactic acid, acetic acid or thioglycolic acid;
[15] A cancer immunotherapeutic agent comprising a TLR2 agonist or TLR4 agonist and a low molecular weight monocarboxylic acid compound;
[16] The cancer immunotherapeutic agent according to [15], wherein the TLR2 agonist is bacterial lipopeptide, peptidoglycan or BCG-CWS, and the TLR4 agonist is LPS;
[17] The cancer immunotherapeutic agent according to [15] or [16], wherein the low molecular weight monocarboxylic acid compound is L-lactic acid, D-lactic acid, acetic acid or thioglycolic acid;
[18] The cancer immunotherapeutic agent according to any one of [15] to [17], further comprising an antigen;
[19] The cancer immunotherapeutic agent according to any one of [15] to [17], further comprising GM-CSF;
About.

  INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an immunostimulatory promoter comprising a low molecular weight monocarboxylic acid compound such as L-lactic acid as an active ingredient, specifically a drug that enhances the immunostimulatory action of a TLR2 agonist and / or a TLR4 agonist. Became. The present invention makes it possible to enhance the activity of BCG-CWS in cancer immunotherapy using BCG-CWS or the like. Furthermore, according to the present invention, it is possible to provide a treatment method effective in a patient with poor responsiveness to BCG-CWS.

  In the present specification, examples of the low molecular weight monocarboxylic acid compound include low molecular weight monocarboxylic acid compounds having one carboxyl group and having a molecular weight of 200 or less, preferably a molecular weight of 60 to 100. Preferable examples include low molecular weight monocarboxylic acid compounds having 2 to 10 carbon atoms, preferably 2 to 5 carbon atoms, which have one carboxyl group and may be substituted with a hydroxyl group or a mercapto group. Specifically, acetic acid, lactic acid or thioglycolic acid can be exemplified as the low molecular weight monocarboxylic acid compound.

In the present specification, the TLR2 agonist is not particularly limited as long as it is a substance that interacts with TLR2 and activates signal transduction of TLR2. Specifically, a bacterial lipopeptide (B. Henderson et al., Microbiol. Rev. Vol 60, No. 2, p316-341, 1996), peptidoglycan (see K. Takeda et al., Annu. Rev. Immunol. Vol. 21, p335-76, 2003) or BCG-CWS (S. Tsuji et al. Infect Immun. Vol. 68 No. 12 p6883-90, 2000). Here, “activate TLR2 signaling” means that IRAK (IL-1R Associated Kinase) binds to MyD88, which is associated with the intracellular TIR domain of TLR2 via TIRAP (TIR-associated protein), and phosphorylates. Activates IRAK, activated IRAK associates with TRAF6, activates JNK and activates AP-1 with TRAF6, or TRAF6 activates IKK and induces IκB degradation to activate NFκB Represents that.
The TLR4 agonist is not particularly limited as long as it is a substance that interacts with TLR4 and activates signal transduction of TLR4, and specific examples include LPS. Here, “activate TLR4 signaling” means that IRAK (IL-1R Associated Kinase) binds to MyD88, which is associated with the intracellular TIR domain of TLR4 via TIRAP, and IRAK is activated by phosphorylation. And activation of TIRAP associated with TLR4 activates IRAK, and activated IRAK associates with TRAF6 to activate JNK and activate AP-1, or TRAF6 activates IKK and degrades IκB Represents activation of NFκB.

In this specification, “immunostimulation” refers to inducing various cytokines and chemokines by activating NF-κB, AP-1, and IRF, and antigen-presenting molecules in antigen-presenting cells that regulate immune responses And the expression of co-stimulatory molecules increases the activation of immunity. Specifically, IL-23 and / or IL-17 secretion promoting activity can be mentioned.
As used herein, “immunostimulatory promoter” means an agent that enhances the immunostimulatory action such as a TLR2 agonist or a TLR4 agonist.

The immunostimulatory promoter of the present invention can be administered together with a TLR2 agonist or TLR4 agonist, or a cancer antigen protein or cancer antigen peptide, and used as a cancer immunotherapy agent. Alternatively, it can be administered together with a virus or bacterial vaccine and used as an agent for treating / preventing a virus or bacterial infection (vaccine enhancer).
When used as a cancer immunotherapeutic agent, the type of cancer is not particularly limited, acute leukemia, chronic leukemia, malignant lymphoma, brain tumor, lung cancer, liver cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, laryngeal cancer, pharynx Cancer, tongue cancer, stomach cancer, esophageal cancer, colorectal cancer, colon cancer, breast cancer, uterine cancer, ovarian cancer, cervical cancer, prostate cancer, lung cancer, brain tumor, multiple myeloma, bone / soft tissue tumor, skin cancer, etc. Is mentioned. Preferable examples include lung cancer and bladder cancer.
Examples of the microbial infection include microbial infections such as viruses, bacteria, fungi, and protozoa. Examples of microbial infections in the present invention include opportunistic infections such as infections that occur in patients suffering from cancer, acquired immune deficiency syndrome (AIDS), and other acquired immune deficiency conditions associated with aging, etc. Is mentioned.
Specific examples of viral infections include cytomegalovirus infection, herpes virus infection, respiratory polynuclear virus infection, and the like.
Specific examples of bacterial infections include Klebsiella pneumonia and tuberculosis.
Specific examples of the fungal infection include cryptococcus infection and candida.
Specific examples of protozoan infections include toxoplasma infections.

When the immunostimulation promoter of the present invention is used as a medicine, it can be administered parenterally or orally.
Examples of preparations for oral administration include capsules, powders, tablets, granules, fine granules, syrups, solutions, suspensions, etc. Examples of preparations for parenteral administration include, for example, Injection, instillation, eye drops, rectal administration, inhalation, spray (spray, aerosol, or liquid / suspension for spraying cartridges for inhalers or aerators), lotion, gel Ointments, creams, transdermal absorbents, transmucosal absorbents, nasal drops, ear drops, tapes, transdermal patch preparations, poultices, external powders and the like. These preparations are prepared by using conventionally known techniques, and acceptable ordinary carriers, excipients, binders, lubricants, stabilizers, disintegrants, buffers, solubilizers, isotonic agents, Surfactants, preservatives, fragrances and the like can be contained, and two or more additives for preparation can be appropriately used.
Among the preparations for oral administration, liquid preparations such as emulsions and syrups are water; sugars such as sucrose, sorbit and fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil and soybean oil; It can be produced using preservatives such as p-hydroxybenzoic acid esters; additives for pharmaceutical preparations such as flavors such as strawberry flavor and peppermint flavor. Solid preparations such as capsules, tablets, powders and granules include lactose, glucose, sucrose, mannitol excipients; disintegrants such as starch and sodium alginate; lubricants such as magnesium stearate and talc; polyvinyl alcohol , Hydroxypropylcellulose, gelatin and other binders; fatty acid esters and other surfactants; glycerin and other plasticizers.
The content of the immunostimulation accelerator of the present invention in the pharmaceutical composition is not particularly limited. For example, in the case of a parenteral preparation, it may be formulated so as to be usually 0.01 mg to 1000 mg, preferably 0.1 mg to 300 mg, more preferably 1 mg to 150 mg per administration.

Among the preparations for parenteral administration, liquid preparations such as injections and drops are preferably prepared as sterilized isotonic liquid preparations. For example, injections can be prepared using an aqueous medium consisting of a salt solution, a glucose solution, or a mixture of saline and glucose solution. Rectal administration agents can be prepared usually in the form of suppositories using a carrier such as cacao butter.
In addition, the immunostimulation accelerator of the present invention itself may serve as an excipient.

The administration method of the immunostimulatory promoter of the present invention may be either oral administration or parenteral administration, but parenteral administration is preferred. In the case of parenteral administration, subcutaneous injection, continuous subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intramuscular injection, intraperitoneal administration, transdermal administration, transmucosal administration, nasal administration and the like can be mentioned. Preferably, intradermal injection is used.
The immunostimulatory promoter of the present invention is usually used together with an immunostimulator such as a TLR2 agonist or a TLR4 agonist, but each component may be administered in the same administration method (administration route) or in different administration methods (administration route). ). The frequency of administration is not particularly limited, but it is usually administered once every several days to several months, or daily administration with a drug holiday of several days to several months.
TLR2 agonists include bacterial cell wall skeletal components (bacteria-CWS), bacterial lipopeptides, or peptidoglycans. These derived bacteria are not particularly limited, and examples thereof include Gram-positive bacilli Mycobacterium bacteria, Nocardia bacteria, Corynebacterium bacteria, Rhodococcus bacteria, and Gordona bacteria. “Mycobacterium spp.” Represents a mycobacteria bacterium that is an acid-fast bacterium, and specifically includes Mycobacterium tuberculosis, Mycobacterium bovis [Mycobacterium bovis, BCG (Including Bacille Calmette-Guerin), Mycobacterium africanum (African fungus), Mycobacterium microti (Mycobacterium tuberculosis), Mycobacterium leprae (Rye fungus), non-tuberculous mycobacteria group Mycobacterium kansasii, Mycobacterium avium, Mycobacterium phlei and the like. Among them, preferred are BCG bacterium, which is a kind of Mycobacterium bovine tuberculosis, and Nocardia rubra, which is a kind of Nocardia group bacteria.
Examples of TLR4 agonists include LPS, monophosphoryl lipid A, and heat shock protein (HSP). LPS-derived bacteria are not particularly limited and include Gram-negative bacteria. Specifically, Escherichia coli (Escherichia coli), Salmonella Minnesota (Salmonella), etc. can be mentioned. The origin of HSP is not particularly limited and includes mammals, Helicobacter bacteria, Mycobacterium bacteria, and the like. Specific examples include mammalian HSP70, HSP22 or HSP60, Helicobacter HSP60, or mycobacterial HSP65.
For example, when used together with BCG-CWS, it is preferable to administer BCG-CWS, 5 to 200 μg at a frequency of once every 1 to 4 weeks, 4 times or more. The dose, the number of administrations, and the administration frequency are appropriately adjusted according to the patient's biological reaction. Specifically, for example, a pharmaceutical composition containing BCG-CWS (5-200 μg) and the immunostimulation promoter of the present invention can be administered four or more times at a frequency of once every 1 to 4 weeks. .

The immunostimulation promoter of the present invention can be used in any of the following forms:
(A) A form of a combination preparation (mixture) obtained by mixing with an immunostimulant that is a TLR2 agonist or a TLR4 agonist.
(B) The form of the kit containing the chemical | medical agent which contains individually the substance which concerns on each said combination.
(C) A combined form in which drugs containing the substances according to each of the above combinations are administered simultaneously, separately or over time.

  When the immunostimulation accelerator of the present invention is a compounding agent (mixture), the compounding ratio with an immunostimulating agent such as BCG-CWS is not particularly limited. What is necessary is just to contain so that each active ingredient may be administered with the said dosage per administration. The compounding agent may be formulated as a compounding agent, or may be prepared (mixed) at the time of administration to a patient.

When the immunostimulation promoter of the present invention is a kit, the immunostimulant is formulated separately and enclosed in separate containers. A kit is a form in which containers containing these respective drugs are present in one packing container.
Each drug (each component) contained in the kit can be prepared (mixed) and used at the time of administration to a patient. Moreover, each chemical | medical agent (each component) contained in a kit can also be used simultaneously, separately, or temporally (for details, refer to the following combination form).

In the case of the combined use in which the immunostimulatory promoter and the immunostimulant of the present invention are administered simultaneously, separately, or over time, each component is prepared separately and enclosed in separate containers. The administration method (administration route such as subcutaneous injection, intradermal injection, intravenous injection) when these are administered simultaneously, separately, or over time may be the same administration method or different administration methods.
When administered separately or over time, any of them may be earlier and later, and the number of administration of each substance is not particularly limited. The administration interval may be immediately after administration of the previous preparation or may be after about 1 day to 1 week.
It is preferable that the immunostimulation promoter and the immunostimulant of the present invention are administered simultaneously.

The cancer immunotherapeutic agent of the present invention can be used together with an antigen protein or an antigen peptide derived from the antigen protein. That is, a pharmaceutical composition containing a low molecular weight monocarboxylic acid compound, a TLR2 agonist or a TLR4 agonist, and an antigen protein or antigen peptide is also within the scope of the present invention.
Here, the antigen protein and the antigen peptide are not particularly limited as long as it is a conventionally known antigen protein and antigen peptide. Specific examples include cancer antigen proteins and cancer antigen peptides derived therefrom, or virus antigen proteins and virus antigen peptides derived therefrom.

Examples of cancer antigen proteins include MAGE (Science, 254: p1643 (1991)), gp100 (J. Exp. Med., 179: p1005 (1994)), MART-1 (Proc. Natl. Acad. Sci. USA, 91: p3515 (1994)), tyrosinase (J. Exp. Med., 178: p489 (1993)), MAGE-related protein group (J. Exp. Med., 179: p921 (1994)), β-catenin (J Exp.Med., 183: p1185 (1996)), CDK4 (Science, 269: p1281 (1995)), HER2 / neu (J.Exp.Med., 181: p2109 (1995)), mutant p53 (Proc Natl.Acad.Sci.USA, 93: p14704 (1996)), CEA (J.Natl.Cancer.Inst., 87: p982 (1995)), PSA (J.Natl.Cancer.Inst., 89: p293) (1997)), WT1 (Proc. Natl. Acad. Sci. USA, 101: p13885 (2004)), HPV (J. Immunol., 154: p5934 (1995)), EBV (Int. Immunol., 7: p653). (1995)).
Examples of the viral antigen protein include antigen proteins derived from viruses such as HIV, hepatitis C virus, hepatitis B virus, influenza virus, HPV, HTLV, and EBV.

  Moreover, as an antigen peptide (peptide vaccine), the partial peptide which consists of an amino acid of about 10 residues of the said antigen protein can be mentioned. Examples of specific antigen peptides are shown below (each number indicates a position on the amino acid sequence of the cancer antigen protein):

(1) Cancer antigen peptide derived from cancer antigen
MAGEA3 peptide 168-176 (Coulie PG et al., Immunol. Rev. 188: 33 (2002)), gp100 peptide 209-217 (Rosenberg SA et al., Nat. Med. 4: 321 (1998)), Melan- A peptide 27-35 (Cormier JN et al., Cancer J. Sci. Am. 3:37 (1997)), Melan-A peptide 26-35, Tyrosinase peptide 1-9, Tyrosinase peptide 368-376, gp100 peptide 280 -288, gp100 peptide 457-466 (Jager E et al., Int. J. Cancer 67:54 (1996)), HER-2 peptide 369-384, HER-2 peptide 688-703, HER-2 peptide 971- 984 (Knutson KL et al., J. Clin. Invest. 107: 477 (2001)), MAGE-A12 peptide 170-178 (Bettinotti MP et al., Int. J. Cancer 105: 210 (2003)), p100 Peptide 280-288 (Phan GQ et al., Proc. Natl. Acad. Sci. USA 100: 8372 (2003))

(2) Virus antigen peptides derived from virus antigens Influenza matrix protein peptide 58-66 (Jager E et al., Int. J. Cancer 67:54 (1996)), HPV16 E7 peptide 86-93 (van Driel WJ et al ., Eur. J. Cancer 35: 946 (1999)), HPV E7 peptide 12-20 (Scheibenbogen C et al., J. Immunother 23: 275 (2000)), HPV16 E7 peptide 11-20 (Smith JWI et al. ., J. Clin. Oncol. 21: 1562 (2003)).

The antigenic protein can be prepared by cloning a cDNA encoding the antigenic protein and expressing it in a host cell according to a basic book such as Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratoy Press (1989).
The synthesis of the antigen peptide can be performed according to a method used in ordinary peptide chemistry. Examples of the synthesis method include literature (Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York, 1976; peptide synthesis, Maruzen. (Inc.), 1975; Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd., 1985; Development of Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991).

  Here, the antigen protein or the antigen peptide can be administered simultaneously or at a different timing from the immunostimulation promoter of the present invention.

  The immunostimulation promoter of the present invention may further contain GM-CSF. GM-CSF can be administered by intravenous injection or intramuscular injection as a solution together with TLR2 or 4 agonist and the immunostimulation promoter of the present invention.

  The present invention will be specifically described in the following examples, but the present invention is not limited thereto.

Example 1
P19 Secretion Promoting Activity by CADO-LC10 Cell Culture Supernatant Cell line CADO-LC10 cells established from lung cancer patients were cultured in DMEM (high glucose) containing 10% FCS, and the culture supernatant was collected. The collected culture supernatant is treated with 100 μg / ml proteinase K at 37 ° C for 1 hour, then treated with 1 mM PMSF to inactivate proteinase K, and 30 kDa or more using an ultrafiltration membrane (Microcon, Milliore) And a fraction of 30 KDa or less. BCG-CWS and each fraction were allowed to act on normal human peripheral blood mononuclear cells, and the mRNA levels of p40 and p19 were measured by real-time PCR. The results are shown in FIG.
As a result, the factor that enhances the expression of p40 was a protein component sensitive to proteinase K in the fraction of 30 KDa or more. On the other hand, it was revealed that the factor that enhances the expression of p19 is a non-protein component resistant to proteinase K at 30 KDa or less.

Example 2
Production of p40 by GM-CSF
CADO-LC10 cell culture components contain about 3.6 units / ml of GM-CSF, and it is known that TLR expression is increased by the action of GM-CSF. Therefore, in order to confirm whether the enhanced expression of p40 is due to the action of GM-CSF, 5 μg of anti-GM-CSF receptor mouse monoclonal antibody (S-20, Santa Cruz Biotechnology) was present in the presence of 10 μg / ml. Peripheral blood mononuclear cells were stimulated with / CG of BCG-CWS, and the expression level of p40 was measured with an ELISA kit. The results are shown in FIG. As a result, the expression of p40 was remarkably suppressed by the anti-GM-CSF receptor antibody. Therefore, the substance that induces the production enhancement of p40 secreted from CADO-LC10 cells was considered to be GM-CSF.

Example 3
Production of p19 by small molecules produced by CADO-LC10 cells
In order to clarify whether the active small molecule produced by CADO-LC10 cells promotes the transcription of p19 gene induced by BCG-CWS stimulation, 6.6 kbp upstream of IL-23p19 gene was transferred to pGL3 promoter vector (promega ) And the G418 resistance gene expression vector were introduced into the J774.1 cell line known to secrete IL-23 by BCG-CWS stimulation to obtain a cell line. The cell line was stimulated with BCG-CWS in the presence of CADO-LC10 cell culture supernatant, and the amount of IL-23 secreted was measured with an ELISA kit (eBioscience). The results are shown in FIG. As a result, the presence of the low molecular fraction in the culture supernatant of CADO-LC10 cells markedly enhanced the secretion of IL-23 as compared to the absence. Moreover, when the luciferase activity in the cell was measured, the activity increased similarly.

Example 4
Immunostimulatory activity of L-lactic acid
The enhancement factor was expected to be L-lactic acid secreted from CADO-LC10 cells because CADO-LC10 cells cultured in a medium with a high glucose concentration had higher enhancement activity. Therefore, in order to confirm whether the small molecule having the enhancing activity contained in CADO-LC10 cells is L-lactic acid, L-lactic acid at the concentration shown in the graph was added to the normal culture solution, and BCG-CWS 10 μg The luciferase assay was performed after stimulation with / ml. The results are shown in FIG. As a result, the luciferase activity increased depending on the concentration of L-lactic acid. The concentration of L-lactic acid contained in the culture supernatant of CADO-LC10 cells was 14 mM, which almost coincided with the value obtained by adding 15 mM L-lactic acid. In addition, when CADO-LC10 cells were treated with an L-lactate dehydrogenase inhibitor (Oxamic acid; oxamic acid) to suppress the production of L-lactic acid, the enhancement activity by the culture supernatant of CADO-LC10 cells was suppressed. It was. Therefore, it became clear that the active low molecular weight substance contained in CADO-LC10 cells is L-lactic acid.

Example 5
Data on the promotion of p19 mRNA level by L-lactic acid
In order to examine whether L-lactic acid actually acts on human peripheral blood monocytes and increases the expression of IL12 / 23p40 subunit and IL23p19 subunit, in the presence of L-lactic acid at the concentration shown in the graph Then, human peripheral blood mononuclear cells were stimulated with 10 μg / ml BCG-CWS for 4 hours, and the mRNA level of IL12 / 23 common subunit p40 or IL23p19 was measured by real-time PCR. The results are shown in FIG. As a result, the expression level of p40 was not enhanced by L-lactic acid, but the expression level of p19 increased depending on the concentration of L-lactic acid. Therefore, the phenomenon shown in the reporter assay was also demonstrated in human cells.

Example 5
Promoting immunostimulation of low molecular weight monocarboxylic acids
Example 4 demonstrates that L-lactic acid works in concert with BCG-CWS stimulation and enhances the production of the IL23p19 subunit. L-lactic acid is a kind of monocarboxylic acid having one carboxyl group. Therefore, we analyzed whether other monocarboxylic acids (acetic acid, thioglycolic acid) had an enhancing effect. In addition, since it may be important that L-lactic acid is an acid, the effect of hydrochloric acid was also examined. When BCG-CWS (10μg / ml) and monocarboxylic acid were allowed to act on J774.1 cells with IL23p19 promoter region (6.6kbp) at various concentrations, acetic acid, thioglycolic acid, and D-lactic acid Although any monocarboxylic acid showed an effect, hydrochloric acid (HCl) showed no enhancement effect. Therefore, the enhancement effect by L-lactic acid was also observed with other monocarboxylic acids, but it became clear that it was not due to acidity alone.

Example 6
L-lactic acid and GM-CSF cooperative action Similar to mouse cells, whether or not L-lactic acid can be enhanced against human monocytes in the presence of 15 mM L-lactic acid, peptidoglycan (PGN) 10 μg / ml Then, human monocyte cells were stimulated, and the amount of IL-23 secretion after 24 hours was measured with an ELISA kit (Bender Medsystems). IL-23 consists of the p19 subunit and the common subunit p40 of IL-12, but since the effect of L-lactic acid is mainly enhanced specifically against p19, the secretion amount of IL-23 protein is Compared with the experiment using a mouse macrophage-derived cell line, it was very small. Thus, it was found that the effect of L-lactic acid works synergistically by coexisting with GM-CSF which induces the production of p40 by increasing the expression of TLR2.

Example 7
Data for various immunostimulants
Various TLR2 or TLR4 agonists were allowed to act on J774.1 cells into which a luciferase gene having a 6.6 kb upstream region of the IL-23p19 gene as a promoter was introduced, and the effects of L-lactic acid were observed. When stimulated with BCG-CWS (TLR2 and TLR4), peptidoglycan (TLR2), Pam3CSK4 (TLR2), and LPS (TLR4), the effect of L-lactic acid was observed with any agonist.

Example 8
Experiments with obabmin peptides
IL-23 induces IL-17 production when acting on memory T cells and Th17 cells, which are effector cells, together with antigen stimulation. Therefore, in order to confirm whether L-lactic acid works in this system, a transgenic mouse having a T cell receptor that specifically recognizes the obabmin peptide (Ovalbumin peptide; OVA323-339) presented in MHC class II. Spleen cells were stimulated with BCG-CWS and L-lactic acid in the presence of obabmin peptide. As a result, IL-17 production was significantly increased by co-stimulation with L-lactic acid compared to BCG-CWS stimulation. This IL-17 production was suppressed by a specific antibody against IL-23. On the other hand, production of interferon γ by the action of BCG-CWS was not changed by L-lactic acid, and was not suppressed by an antibody against IL-23. Therefore, it was considered that the effect of L-lactic acid works in conjunction with BCG-CWS stimulation and enhances IL-23-IL-17 pathway by enhancing IL-23 secretion.

Example 9
Anti-tumor effect
The effect of L-lactic acid on the anticancer effect of BCG-CWS was examined using a B16 melanoma transplantation model. Mice received 6 × 10 ⁵ B16 melanoma cells subcutaneously. At 12, 15, 18, and 21 days after tumor implantation, 2.5 × 10 ⁵ B16 melanoma cells, frozen and thawed as antigen, alone or with 20 μg BCG-CWS or with 20 μg BCG-CWS and final concentration Intradermal administration with 20 mM L-lactic acid {In FIG. 10, antigen only (closed diamond, T--), antigen and BCG-CWS (closed circle, TB-), antigen and BCG-CWS and L-lactic acid ( open circle, TBL)}. The size of the tumor was 1 on the 11th day after tumor administration. As a result, there was no significant difference between BCG-CWS and antigen, but by adding L-lactic acid, tumor growth was significantly suppressed at p <0.05 on the 16th, 18th and 19th day.

  The immunostimulation promoter of the present invention can be used as a medicine.

FIG. 3 is a diagram showing the secretion promoting activity of p19 by CADO-LC10 cell culture supernatant divided into a fraction having a molecular weight of 30 KDa or less and a fraction having a molecular weight of 30 KDa or more. The vertical axis shows the value obtained by correcting the mRNA amount of p40 and p19 with the mRNA amount of β-actin (actin). The fraction of 30 KDa or more showed p40 expression promoting activity, but the activity was lost by proteinase K. On the other hand, the fraction of 30 KDa or less showed p19 expression promoting activity, and it was found that the activity was not changed by proteinase K. It is the figure which showed that GM-CSF accelerates | stimulates production of p40 of IL-12 / 23. The p40 production promoting activity by LC-10 culture supernatant was offset by anti-GM-CSFR antibody. From this, it was found that the p40 production promoting activity is due to GM-CSF. It is a figure which shows the gene for reporter gene assays for measuring p19 expression promotion activity. The left figure is an ELISA result showing that CADO-LC10 cell culture supernatant increases the production of IL-23 protein by BCG-CWS. The right figure shows that BCG-CWS acts on the promoter of p19 to promote the expression of p19, and the CADO-LC-10 cell culture supernatant enhances the expression promoting activity of BCG-CWS. It is a figure which shows that L-lactic acid increases the expression promoting activity of p19 by BCG-CWS in a concentration-dependent manner. The left figure shows that the p40 expression promoting activity by BCG-CWS is not affected by L-lactic acid. The right figure shows that the p19 expression promoting activity by BCG-CWS is enhanced by L-lactic acid in a concentration-dependent manner. It is a figure which shows the effect which other low molecular weight monocarboxylic acid has on the p19 expression promotion activity by BCG-CWS. The horizontal axis represents the concentration of low molecular weight monocarboxylic acid, and the vertical axis represents luciferase activity (p19 expression promoting activity). It is a figure which shows the synergistic increase of IL-23 production promotion activity of peptidoglycan by using together GM-CSF and L-lactic acid. It is a figure which shows that the p19 expression promotion activity of various TLR2 or 4 agonist is enhanced by L-lactic acid. It is a figure which shows the experimental result of IL-17 production promotion effect of BCG-CWS using an obabmin peptide. The left figure shows that L-lactic acid does not affect the IFN-γ production promoting activity of BCG-CWS. The right figure shows that L-lactic acid enhances IL-17 production promoting activity of BCG-CWS, which is offset by anti-IL-23 antibody. It is a figure which shows that L-lactic acid increases the anti-tumor effect of BCG-CWS. “*” Indicates a significant difference (p <0.05) between the group administered with the antigen, BCG-CWS and L-lactic acid, and the group administered with only the antigen.

Claims (19)

An immunostimulation accelerator containing a low molecular weight monocarboxylic acid compound as an active ingredient. The immunostimulation accelerator according to claim 1, wherein the low molecular weight monocarboxylic acid compound is L-lactic acid, acetic acid or thioglycolic acid. The immunostimulation promoter according to claim 1 or 2, which is an IL-23 and / or IL-17 secretion promoter. The immunostimulation promoter according to any one of claims 1 to 3, wherein the immunostimulation is performed by a TLR2 agonist and / or a TLR4 agonist. The immunostimulation promoter according to claim 4, wherein the TLR2 agonist is bacterial lipopeptide, peptidoglycan or BCG-CWS, and the TLR4 agonist is LPS. The immunostimulation promoter according to any one of claims 1 to 3, wherein the immunostimulation is caused by a cancer antigen. The immunostimulation promoter according to any one of claims 1 to 3, wherein the immunostimulation is by a microorganism or a microbial vaccine. The immunostimulation promoter according to any one of claims 1 to 6, which is a cancer immunotherapy agent. The immunostimulation promoter according to any one of claims 1 to 5 and 7, which is a microbial infection therapeutic agent or a preventive agent. The immunostimulation promoter according to claim 9, wherein the microbial infection is an opportunistic infection. The immunostimulation promoter according to claim 10, wherein the opportunistic infection is Klebsiella pneumonia, cryptococcus infection, tuberculosis, toxoplasma infection, or Candida infection. A TLR2 agonist and / or TLR4 agonist activity enhancer comprising a low molecular weight monocarboxylic acid as an active ingredient. The TLR2 agonist and / or TLR4 agonist activity enhancer according to claim 12, wherein the TLR2 agonist is bacterial lipopeptide, peptidoglycan or BCG-CWS, and the TLR4 agonist is LPS. The TLR2 agonist and / or TLR4 agonist activity enhancer according to claim 12 or 13, wherein the low molecular weight monocarboxylic acid compound is L-lactic acid, acetic acid or thioglycolic acid. A cancer immunotherapeutic agent comprising a TLR2 agonist or a TLR4 agonist and a low molecular weight monocarboxylic acid compound. The cancer immunotherapeutic agent according to claim 15, wherein the TLR2 agonist is bacterial lipopeptide, peptidoglycan or BCG-CWS, and the TLR4 agonist is LPS. The cancer immunotherapeutic agent according to claim 15 or 16, wherein the low molecular weight monocarboxylic acid compound is L-lactic acid, D-lactic acid, acetic acid or thioglycolic acid. The cancer immunotherapeutic agent according to any one of claims 15 to 17, further comprising an antigen. The cancer immunotherapeutic agent according to any one of claims 15 to 17, further comprising GM-CSF.

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