WO2008056705A1 - Anticorps spécifique des cellules m - Google Patents

Anticorps spécifique des cellules m Download PDF

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Publication number
WO2008056705A1
WO2008056705A1 PCT/JP2007/071645 JP2007071645W WO2008056705A1 WO 2008056705 A1 WO2008056705 A1 WO 2008056705A1 JP 2007071645 W JP2007071645 W JP 2007071645W WO 2008056705 A1 WO2008056705 A1 WO 2008056705A1
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Prior art keywords
cells
antibody
antigen
epithelium
fae
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PCT/JP2007/071645
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English (en)
Japanese (ja)
Inventor
Hiroshi Kiyono
Yoshikazu Yuki
Osamu Igarashi
Tomonori Nochi
Kazutaka Terahara
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The University Of Tokyo
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Priority to JP2008543105A priority Critical patent/JPWO2008056705A1/ja
Publication of WO2008056705A1 publication Critical patent/WO2008056705A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies

Definitions

  • the present invention relates to an antibody having high specificity for M cells or lymphoid follicular epithelium (FAE), which is a tissue surrounding M cells.
  • FAE lymphoid follicular epithelium
  • the invention also relates to the use of such antibodies as vaccine carriers.
  • One of the routes of entry of antigens such as pathogenic microorganisms and allergens into the living body is the surface of mucosal tissues including the oral cavity, digestive tract and respiratory organs.
  • SARS virus, HIV virus, Ebola virus, hepatitis B virus, Bacillus anthracis, plague fungus, Q fever rickettsia, etc. are known as pathogenic microorganisms that infect through such mucosal tissue surface (mucosal surface).
  • the mucosal surface is constantly under attack as a site of entry of many of these pathogenic microorganisms from the outside world.
  • the mucosal immune system is a recognition / response system for foreign antigens including these pathogenic microorganisms.
  • the mucosal immune system is not only a humoral immune response centered on antigen-specific IgG in the whole body serum, but also on the mucosal surface against foreign foreign antigens taken in through the mucosal surface. Induces a humoral immune response centered on the secretory immunoglobulin A (S-IgA), and also induces a cellular immune response centered on mucosal cytotoxic T cells (CTLs) It has been known.
  • S-IgA secretory immunoglobulin A
  • CTLs mucosal cytotoxic T cells
  • Peyer's Patch which is classified as Gut-Associated Lymphoid Tissue (GALT) plays an important role in the immunocompetent tissue involved in the IgA immune response that is the center of mucosal immunity in the gastrointestinal tract. do.
  • FAE Food Associated Epithelium
  • Mfold microfold cells
  • Antigen is taken into Peyer's patch from the intestinal lumen side.
  • the antigen-specific immune response is triggered by the foreign antigen taken up from these M cells being delivered to antigen-presenting cells that are located in recesses (pockets) on the M cell basement membrane side.
  • M cells are thought to have an important role as an initiator in the mucosal immune system.
  • the antigen thus taken up is processed by antigen-presenting cells such as macrophages and dendritic cells, and as a result of antigen presentation, IgA-producing progenitor B cells are induced and IgA antibody production occurs.
  • mucosal vaccines that induce mucosal immunity via the intestinal tract do not require injection, and are not only safe and simple, but also mucosal systems and whole body. It is considered to be an ideal vaccine in terms of establishing antigen-specific protective immunity in both immune systems of the system! In inducing an effective immunogen (antigen) -specific intestinal mucosal immune response, how vaccine antigens can be effectively targeted to small intestinal mucosa-derived tissues, especially M cells, which are specialized in antigen uptake. It is considered a key.
  • Non-patent Document 1 a DNA vaccine using the M cell targeting method has been reported as a method for effectively inducing mucosal immunity.
  • This method uses a reovirus protein ⁇ 1 that has the ability to bind to M cells to deliver vaccines to sputum cells.
  • ⁇ 2-3 type sialic acid which is a receptor for ⁇ 1 protein, is present not only in sputum cells but also in small intestinal epithelial cells. Technology development was essential.
  • Non-patent Document 2 lectin UEA-1 has been known as a marker for recognizing mouse sputum cells.
  • UEA-1 is also reactive with goblet cells, which are mucus-secreting cells that are not only sputum cells (Non-Patent Document 2). Therefore, UEA-1 is used to administer vaccine antigens.
  • it is difficult not only to target goblet cells but also to goblet cells and to elicit an immune response effectively.
  • Non-Patent Document 1 Wu Y et al. Proc. Natl. Acad. Sci. USA 2001, 98: 9318-23
  • Non-Patent Document 2 Kandori, H. et al., Exp. Anim. (1996) 45: 155-160
  • the inventors of the present application provide an epithelial layer of an M cell that hits an antigen uptake port or its peripheral portion.
  • the present inventors have conducted extensive research for the purpose of developing a molecule that specifically binds to the lymphoid follicular epithelial layer (FAE) of the M cell or its peripheral part.
  • the inventors have found that the above-mentioned problems can be solved by preparing an antibody capable of targeting the lymphoid follicular epithelial layer (FAE) in the surrounding area.
  • the present invention relates to (1) an antibody that recognizes all or part of lymphoid follicular epithelium (FAE) but does not recognize chorionic epithelium and glandular epithelium, or (2) M cells. Recognizing power Does not recognize goblet cells, absorptive epithelial cells and respiratory epithelial cells!
  • FAE lymphoid follicular epithelium
  • the complex of this antibody and the immunogen (antigen) administered orally is specifically transported to or near the surface of the M cell, which is an immunogen (antigen) uptake cell in the mucosa. .
  • the immunogen (antigen) is easily taken up from M cells, and mucosal immunity can be specifically and efficiently induced against the target immunogen (antigen).
  • mucosal immunity is specifically and efficiently induced against the target immunogen (antigen)
  • the pathogenic microorganism is neutralized by neutralizing the target pathogenic microorganism (or pathogenic toxin). Diseases caused by substances (or pathogenic toxins) can be prevented or treated.
  • the present invention also provides a vaccine carrier for mucosal vaccines comprising the above-described antibody.
  • the antibody of the present invention recognizes all or part of lymphoid follicular epithelium (FAE), but does not recognize chorionic epithelium and glandular epithelium, or detects M cells. The ability to recognize S, goblet cells, absorptive epithelial cells and respiratory epithelial cells are not recognized. Due to this feature, when this antibody is bound to an immunogen (antigen) and administered orally, the immunogen (antigen) becomes an immunogen (antigen) in the mucosa based on the specific binding between the antibody and M cells.
  • the antibody of the present invention can be used as a vaccine carrier for a mucosal vaccine by being combined with an immunogen (antigen) because it is specifically transported to the vicinity of M cells that are uptake cells.
  • FIG. 1 shows a stained image with # 16-2-4 antibody (green fluorescence, 16-2-4), lectin UEA-1 A stained image (red fluorescence, UEA-1) and a superimposed image (Merge) thereof are shown.
  • Fig. 2 shows the staining image of the # 17-1-8-4 antibody (green fluorescence, 17-1-8-4), lectin UEA- 1 shows a stained image (red fluorescent light, UEA-1) and a superimposed image (Merge) thereof.
  • Fig. 3a shows a nuclear staining image by DAPI (blue fluorescence, DAPI) as a counterstained color when immunohistological analysis was performed on Peyer's patches of the small intestine, and staining with # 16-2-4 antibody Images (green fluorescence, 16-2-4), stained images with lectin UEA-1 (red fluorescence, UEA-1), and their superimposed images (Merge) are shown.
  • DAPI blue fluorescence, DAPI
  • FIG. 4 is a diagram showing the results of immunoelectron microscopic analysis using antibody # 16-2-4.
  • the right figure is an enlarged view of the part enclosed by the square in the left figure.
  • FIG. 5a is a stained image of a nasopharyngeal tissue section of the upper respiratory tract, and the portion indicated by an arrow is nasopharyngeal associated lymphoid tissue (NALT).
  • Fig. 5b shows a stained image (green fluorescence, 16-2) when immunohistological analysis was performed on the boxed area (NALT) in Fig. 5a (NALT).
  • -4 stained image with lectin UEA-1 (red fluorescence, UEA-1), nuclear stained image with DAPI (blue fluorescence, DAPI) and counter-stained image (Merge).
  • FIG. 6 shows that FITC-labeled # 16-2-4 antibody and FITC-labeled control antibody (Rat IgG) were administered into the intestinal loop containing Peyer's patch and the subsequent kinetics of the antibody was observed histologically.
  • Figure 6A and 6B show the results of the follow-up evaluation.
  • Fig. 7 shows the combination of # 16-2-4 antibody and tetanus toxoid antigen (16-2-4 T
  • Fig. 8 shows the combination of # 16-2-4 antibody and botulinum toxoid antigen (16-2-4
  • Fig. 9 shows the combination of # 16-2-4 antibody and botulinum toxoid antigen.
  • FIG. 5 is a view showing an antigen neutralizing effect against botulinum toxin by an antibody-antigen complex.
  • an antibody or M cell that recognizes all or part of the lymph follicle epithelium (FAE) but does not recognize the villi and glandular epithelium is used. It recognizes but does not recognize goblet cells, absorptive epithelial cells and respiratory epithelial cells! / And provides an antibody.
  • FAE lymph follicle epithelium
  • Lymphoid follicular epithelium refers to the epidermis part of the lymphoid follicle with a dome-like structure that covers the nozzle plate and exists in the intestine and nasopharynx.
  • This lymphoid follicular epithelium (FAE) is known to exhibit different properties from the villous epithelium in the normal intestinal tract or the glandular epithelium in the nasopharynx. It is composed of M cells and respiratory epithelial cells!
  • the main function of the intestinal villous epithelium is the absorption of nutrients and the function of mucus secretion, so it is mainly composed of absorption epithelial cells and goblet cells.
  • the main function of the glandular epithelium is the discharge of foreign matter from the nasopharynx, which absorbs nutrients. It is composed of respiratory epithelial cells and goblet cells.
  • M cells are special cells specialized in antigen uptake that exist in intestinal tract-related lymphoid tissues (GALT) such as Peyer's patches and nasopharyngeal-related lymphoid tissues (NALT) in the upper respiratory tract.
  • GALT intestinal tract-related lymphoid tissues
  • NALT nasopharyngeal-related lymphoid tissues
  • transcytosis an intracellular transport system called transcytosis, the intestinal antigen or airway antigen is actively taken up, and the antigen taken up by antigen-presenting cells such as macrophages or dendritic cells present in Peyer's patches or NALT is received. Passing it is thought to play a major role in triggering immune surveillance.
  • Mouse M cells are generally characterized by having a marker that binds to the lectin U EA-1. However, until now there has been no marker molecule that can recognize only M cells, so the microvilli have a rough and pocket structure to identify M cells. Based on the histological characteristics of! So
  • lymphoid follicular epithelium FAE
  • Absorption epithelial cells responsible for nutrient absorption are the most abundant cells in the small intestinal epithelium, and in the nasopharynx, there are many respiratory epithelial cells that do not absorb nutrients.
  • the antibody of the present invention recognizes all or part of the Follicle Associated Epithelium (FAE) but does not recognize the chorionic and glandular epithelium, or recognizes M cells but does not It does not recognize cells, absorptive epithelial cells and respiratory epithelial cells! /, And provides antibodies, so that the entire follicular epithelium (FAE) including M cells or peripheral areas of M cells in Peyer's patches or Some can be specifically recognized.
  • FAE Follicle Associated Epithelium
  • the antibody produced in the present invention may be a polyclonal antibody or a monoclonal antibody as long as it has the above-mentioned binding characteristics.
  • a sample containing M cells or lymphoid follicular epithelium (FAE) as an immunogen is prepared from, for example, an animal Peyer's patch.
  • FAE lymphoid follicular epithelium
  • an immunogen sample is obtained from, for example, a mouse
  • cells are separated by treating the small intestine tissue of the mouse with EDTA, and the cells are stained with lectin UEA-1 and then subjected to UEA-1 using a cell sorter.
  • positive cells Prepare as a sample containing both M cells and goblet cells.
  • the immunogen sample prepared as described above is prepared in accordance with a conventional method from an immunized animal (for example, a mouse, a goat, a rabbit, a mouse, a rat in the case of a polyclonal antibody, and a mouse in the case of a monoclonal antibody. , Rats, etc.) to produce antibodies in the body.
  • an immunized animal for example, a mouse, a goat, a rabbit, a mouse, a rat in the case of a polyclonal antibody, and a mouse in the case of a monoclonal antibody. , Rats, etc.
  • serum from an immunized animal or immune globin purified from the serum can be used.
  • a monoclonal antibody plasma cells derived from the spleen such as mouse rat immunized with the above-described immunogen sample are fused with myeloma cells to produce hyperidoma cells. It is possible to select and obtain clones that produce antibodies that react with all or part of M cells or FAE. Usually, a monoclonal antibody can be obtained by injecting the hybridoma thus prepared into the peritoneal cavity of a mouse and purifying the immunoglobulin accumulated in the ascites. It is also possible to cultivate hyperidoma cells in a serum-free medium and purify the culture supernatant.
  • mouse-lat hybridoma cells were prepared by preparing spleen cells that produce monoclonal IgG antibodies using rats and fusing them with the mouse myeloma cell line (AG8-653). From these mouse-rat hybridoma cells, cells having the desired binding characteristics were screened. As a result, mouse-rat hybridoma cells 16-2-4 cells (FERM ABP-10926) and 17-1-8- Four cells (FERM ABP-1092 7) were prepared and their isotypes were determined to be rat IgG2c (# 16-2-4) and rat HgA (# 17-1-8-4).
  • the antibody thus obtained can be used as an antibody for detecting an M cell-specific or FAE-specific marker in a tissue.
  • the antibody of the present invention is also capable of specifically delivering an immunogen (antigen) to the M cell or the FAE region in the vicinity thereof by binding to the target immunogen (antigen) and orally administering the antibody. It also has the feature.
  • the antibody of the present invention can be used as a vaccine carrier. That is, the present invention has the ability to recognize all or part of the lymphoid follicular epithelium (FAE), chorionic epithelium and glandular hair.
  • a vaccine carrier comprising an antibody that does not recognize skin, or an antibody that recognizes M cell-recognizing goblet cells, absorption epithelial cells, and respiration epithelial cells.
  • antibody # 17 -1-8-4 described above as an antibody that recognizes all or part of the above-mentioned lymphoid follicular epithelium (FAE, Follicle Associated Epithelium) but does not recognize chorionic epithelium and glandular epithelium.
  • FAE lymphoid follicular epithelium
  • the above-mentioned antibody # 16-2-4 can be used as an antibody.
  • the vaccine carrier of the present invention can be used as an auxiliary for transmucosal administration of an immunogen bound to an antibody via M cells.
  • the term “mucosa” refers to a mucosa in which M cells are present, and is a general term for a portion covering a lumen surface of a luminal organ such as a digestive organ or a respiratory organ. It is covered with mucus secreted from goblet cells and physically prevents the invasion of pathogenic microorganisms.
  • mucosa that can be applied to the vaccine carrier of the present invention include nasal cavity, oral cavity, lung, and small intestine.
  • the immunogen By applying an antibody that binds an immunogen to these mucosal surfaces, the immunogen can be delivered to the vicinity of the M cell, and as a result, the immunogen taken up by transcytosis by the M cell, etc. Transported through M cells and taken up by endocytosis by immature dendritic cells in the M cell pocket just below the basement membrane and transported to the parafollicular area, which is the T cell area, where dendritic cells are Matures to become an antigen-presenting cell and activates antigen-specific CD4 + T cells.
  • B cells that have been similarly sensitized are activated by the antigen-specific CD4 + T cells described above.
  • the antigen-vaccine carrier complex is delivered to these mucosal surfaces.
  • Any administration method that can be used may be used. Such administration methods include, but are not limited to, oral administration and nasal administration.
  • an adjuvant may be further included for the purpose of adding immunostimulatory activity.
  • Adjuvants that can be administered together with vaccines using the vaccine carrier of the present invention include detoxified cholera toxin, detoxified Escherichia coli heat-labile toxin, monophosphoryl lipid A, cyto force-in, chemokine , Lectin, saponin and the like.
  • the purpose of this example was to produce a monoclonal antibody specific for the F cell (Follicle Associated Epithelium; FAE), which is the M cell serving as an antigen uptake port or its peripheral part.
  • F cell Follicle Associated Epithelium
  • UEA-1 After that, the isolated cells are stained with UEA-1 and then subjected to a cell sorter (FACSAria, Becton Dickinson) to collect UEA-1-positive cells (0.5 to 1.0 ⁇ 10 6 cells) and adjuvant (TiterMax).
  • TM Gold was immunized intradermally (foot pad) in SD rats.
  • UEA-1 also binds to goblet cells that are not only M cells (Non-Patent Document 2), and this cell group may contain goblet cells in addition to M cells. Estimated.
  • the prepared rat spleen cells were fused according to a conventional method using a mouse myeloma cell line (P3X63-AG8.653) and PEG 1500 (Ro che) to prepare hyperidoma cells.
  • a mouse myeloma cell line P3X63-AG8.653
  • PEG 1500 Ro che
  • the antibodies produced from about 1000 kinds of Hypridoma cell clones thus obtained can bind to M cells or lymphoid follicular epithelium (FAE) in the surrounding area, and From the viewpoint of whether or not it has the characteristic of not binding to cells, immunohistological screening using culture supernatants produced M cell specific antibodies and lymphoid follicular epithelial (FAE) specific antibodies. The presence or absence was evaluated.
  • the inventors of the present invention provide mouse-rat hybridoma cells 16-2-4 cells (FERM ABP-10926) and # 16-2-4 antibodies and # 17-1-8-4 antibodies, respectively, and 17-1-8-4 Cells (FERM ABP-10927) were transferred to the International Patent urganism Depositary (IPOD), National Institute or Avionic Industrial Science and Technology (AIST). )
  • the # 16-2-4 antibody (Rat IgG2c) and the # 17-1-8-4 antibody (Rat IgA) were used for immunohistochemistry. The binding properties of these antibodies were examined.
  • antibody # 16-2-4 reacted specifically with UEA-1-positive M cells (Fig. 1).
  • antibody # 17-1-8-4 showed specificity in Peyer's patch lymphoid follicular epithelium (FAE) and did not react with chorionic epithelium (Fig. 2).
  • the yellow stained cells indicated by the red arrows are M cells, and the red stained cells indicated by the yellow arrows are goblet cells.
  • an immunoelectron microscopic analysis using a colloidal gold (18 nm) -labeled anti-rack HgG antibody (Jackson) as a secondary antibody was performed. The results are shown in Fig. 4. As a result, antibody # 16-2-4 showed high specificity for typical M cells with coarse microvilli and pocket structure, whereas intestinal epithelial cells (IEC) ) was not observed. From this result, it was also shown that the antibody # 16-2-4 binds to M cells with high specificity S, compared with lectin UEA-1.
  • NAL T nasopharyngeal associated lymphoid tissue
  • # 16-2-4 antibody rabbit IgG2c
  • the mouse nasal cavity tissue containing NALT was fixed with 4% paraformaldehyde solution, and a 7 ⁇ -thick slice was prepared according to a conventional method and subjected to histological analysis.
  • FIG. Fig. 5a is a hematoxylin-eosin-stained image of a tissue section, and the part indicated by an arrow is NALT.
  • NALT boxed area in Fig. 5a was stained with # 16-2-4, then stained with FITC-labeled anti-rat Ig's antibody (Biosource) and TRITC-labeled UEA-1 (Vector).
  • TCS SP2, Leica focused laser microscope
  • # 1 6-2-4 antibody reacts specifically with UEA-1-positive M cells. The force showed no responsiveness.
  • a vaccine antigen and an antibody-antigen complex were prepared using antibody # 16-2-4 of the antibodies prepared in Example 1, and this was orally immunized to mice.
  • tetanus toxoid was used as a vaccine antigen and bound to # 16-2-4 antibody by the avidin-biotin method (16-2-4 TT group). Specifically, biotin-labeled tetanus toxoid (50 g) was bound with the same amount (50 g) of avidin, and then reacted with biotin-labeled # 16-2-4 antibody (100, ig). An antibody-antigen complex was prepared.
  • the antibody-antigen complex (200 ⁇ g) was orally administered to Balb mice (7 weeks old, female) together with mucosal adjuvant cholera toxin (Choler a toxin; CT, 10 ⁇ g) (once a week) A total of 2 times).
  • FIG. # Combined 16-2-4 antibody and TT (16-2-4 TT group) and immunized orally, systemic response (serum IgG) (Fig. 7A) and mucosal response (fecal IgA) ( In both cases, high titer TT-specific antibody production was induced (Fig. 7B).
  • TT conjugated with a control antibody is administered orally (Rat IgG-TT group)
  • PBS Group when negative control PBS is administered
  • TT When inducing antibody production to TT administered orally by a conventional method, about 250 ag of TT is frequently orally administered together with 10 g of cholera toxin. According to the method of the present invention, as compared with the usual method, only when TT is bound to the antibody of the present invention, only a very small amount of TT (50 g) is orally administered and specific to TT. It was revealed that not only general systemic response (serum IgG) antibodies but also mucosal response (stool IgA) antibodies can be induced effectively.
  • the antigen of the present invention is bound to the target antigen to be immunized and orally immunized, whereby the antigen is taken up from the M cell based on the specific binding between the antibody and the M cell.
  • the antigen is taken up from the M cell based on the specific binding between the antibody and the M cell.
  • a vaccine antigen and an antibody-antigen complex were prepared using antibody # 16-2-4 of the antibodies prepared in Example 1, and this was orally immunized to mice.
  • botulinus toxoid was used as a vaccine antigen and bound to antibody # 16-2-4 by the avidin-biotin method. Specifically, the same amount (50 g) of avidin is bound to biotin-labeled borinustoxoid (50 g), and then reacted with biotin-labeled # 16-2-4 antibody (10011 g).
  • the antibody-antigen complex was prepared with The antibody-antigen complex (200 ⁇ g) was orally administered to Balb mice (7 weeks old, female) with cholera toxin (CT, 10, 1 g) as a mucosal adjuvant (week 1). 3 times).
  • the antibody of the present invention is bound to the target antigen to be immunized, and oral immunization is performed, whereby the antibody and M cells are specifically identified. Based on the binding, antigens were taken up from M cells and were found to cause mucosal immunity very effectively.
  • a vaccine antigen and antibody-antigen complex was prepared using antibody # 16-2-4 of the antibodies prepared in Example 1, and this was orally immunized to mice. The neutralizing effect against the antibody induced by the sputum cell-targeted mucosal vaccine was evaluated.
  • botulinus toxoid was used as a vaccine antigen and bound to antibody # 16-2-4 by the avidin-biotin method (# 16-2-4-BT group).
  • mice [0063] As a control, normal rat HgG (Sigma) and BT were bound by the same method (Rat IgG-BT group) and orally administered to mice.
  • the antibody of the present invention recognizes all or part of lymphoid follicular epithelium (FAE) but does not recognize chorionic and glandular epithelium, or detects M cells. The ability to recognize S, goblet cells, absorptive epithelial cells and respiratory epithelial cells are not recognized. Due to this feature, when this antibody is bound to an immunogen (antigen) and administered orally, the immunogen (antigen) becomes an immunogen (antigen) in the mucosa based on the specific binding between the antibody and M cells.
  • the antibody of the present invention can be used as a vaccine carrier for a mucosal vaccine by being combined with an immunogen (antigen) because it is specifically transported to the vicinity of M cells that are uptake cells.

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Abstract

Cette invention propose de développer un moyen pour permettre une administration efficace d'un antigène dans des cellules M et ainsi une augmentation remarquable de l'efficacité d'un vaccin muqueux, en ciblant particulièrement la couche de surface de l'épithélium (FAE) des cellules M ou la périphérie de celles-ci à partir de laquelle un agent est incorporé. Il a été découvert que le problème décrit ci-dessus peut être résolu par la construction d'un antigène qui peut cibler l'épithélium associé aux follicules (FAE) des cellules M ou la périphérie de celles-ci.
PCT/JP2007/071645 2006-11-10 2007-11-07 Anticorps spécifique des cellules m WO2008056705A1 (fr)

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