JP2005187428A - Filovirus treating agent by anti-mgl antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for the prevention and/or treatment of viral infectious diseases, especially infectious diseases caused by viruses each having a mucin-type glycoprotein represented by filovirus, and obtain a pharmaceutical composition for the production and treatment method. <P>SOLUTION: The method for the prevention and/or treatment of infectious diseases of the viruses each having the mucin-type (O-bonded type) sugar chain contains a step to inhibit the interaction of a macrophage galactose-type C lectin (MGL) on a macrophage with the mucin-type sugar chain on the virus particle in a patient necessitating the treatment. This invention further provides a pharmaceutical composition useful for the prevention/treatment method. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for treating a viral infection represented by filovirus infection and a pharmaceutical composition for the treatment.

Filovirus infections against filovirus primates show very severe bleeding signs and high mortality compared to those caused by other hemorrhagic fever viruses. In virus Filoviridae causing the infection (Filoviridae), Marburg (Marburg) and two genera are known Ebola (Ebola), further the latter four species, namely, Zaire (Zaire), Sudan (Sudan), Ivory Coast and Reston. Of the four species, Zaire is considered the most highly toxic, and it has been reported that about 90% of infected people died (in the 1976 Zaire (now Congo) and Sudan epidemics) 430 of 500 people died). Regarding the course of infection by the virus, the incubation period after infection is 4 to 16 days, and headache and back pain begin immediately after the onset of illness. High fever, ocular conjunctivitis, pharyngitis, chest pain with cough, etc. occur around the second day after the onset of symptoms, followed by symptoms of vomiting and diarrhea, and sometimes bloody stool to tar stool. The patient rapidly became dehydrated, and bleeding tendency such as bleeding from gingiva, nose, vagina, subcutaneous, etc. from the 5th to 7th days, vomiting, melena, etc. Appears in sex. About half of the patients die from strong toxic symptoms and disseminated intravascular coagulation (DIC) on the 4th to 10th day after onset of illness.

  Against this background, Ebola virus infection, that is, Ebola hemorrhagic fever, is regarded as an international infectious disease that requires strict isolation treatment. Reston species have been reported to be less pathogenic than other Ebola species in non-human primate experiments, and in fact, no symptomatic infection has been confirmed in humans.

  In molecular biology, filoviruses are enveloped non-segmented single-stranded (-) polar RNA viruses that contain at least seven structural proteins. All of these proteins are translated from monocistronic polyadenylated mRNA transcripts. The fourth gene from the 3 ′ end of the filovirus genome encodes an envelope glycoprotein (GP) and is considered to be involved in receptor binding and fusion to the host cell membrane of the virus (Non-patent literature). 1 and 2). From the gene, an envelope GP and a non-structural secretory glycoprotein are expressed (Non-patent Documents 3 and 4). These glycoproteins are highly glycosylated and have an N-linked sugar chain and an O-linked sugar chain (mucin-type sugar chain). Depending on the cell line used, the terminal silylation pattern is different (Non-Patent Document 5).

  Recent studies on the infection of the filovirus have shown that dendritic cell specific ICAM-3 adherent non-Itegrin (DC-SIGN) or liver / lymph node specific ICAM-3 adherent non-Itegrin (L-SIGN) It was clarified that it plays an important role in infection. That is, Non-Patent Documents 6 to 9 show that when DC- or L-SIGN is overexpressed on the cell surface, these molecules recognize highly glycosylated Ebola GP, and the pseudotype of the virus. And showed that it promotes infection with genuine virus. In particular, high mannose-type N-glucan bound to viral GP is considered to be important in the interaction with lectins such as DC-SIGN and L-SIGN (Non-patent Document 10).

MGL
It is known that related cells derived from macrophages and myeloids express various lectins that recognize cell surfaces or soluble glycoproteins such as galectin-3, sialoadhesin, and mannose receptor. It has been. These lectins on macrophages are involved in cell-cell adhesion, hematopoiesis, endocytosis, and cleaning of necrotic cells and molecules. Macrophage galactose type C lectin (MGL) (Non-Patent Document 11) is one of the above-mentioned lectins, and is a type 2 transmembrane protein expressed on macrophages and immature dendritic cells. The inventors of the present invention have previously cloned the lectin in mice (Non-patent Documents 12 and 13). Furthermore, the gene products (including splicing variants) are in vitro, and the end of the galactose terminal glycoprotein is in vitro. It has been reported that it is involved in cytosis (Non-patent Documents 14 and 15).

  Human MGL (hMGL) has also been identified, and this lectin also recognizes a sugar chain having an O-linked galactose / N-acetylgalactosamine (ie, a mucin-type sugar chain) particularly strongly. In humans, this protein is expressed on monocyte-derived immature dendritic cells and macrophages, and has been reported to function as an endocytosis receptor for galactosylated glycoprotein antigens (Non-patent Document 12). And 16).

  The hMGL has so far been confirmed to be the only C-type lectin specific to galactose / N-acetylgalactosamine on macrophages and to exhibit a ligand specificity different from that of the DC-SIGN and L-SIGN. (Non-patent Document 12).

However, there was no direct knowledge regarding the interaction between MGL and viral glycoprotein. In particular, considering the involvement of the above-mentioned DC-SIGN and L-SIGN in the filovirus infection process and the different ligand specificities between them, other lectin molecules, especially MGL molecules may be used in the virus infection process. There was no suggestion of involvement.
Takada, A., C. Robison, H. Goto, A. Sanchez, KG Murti, MA Whitt, and Y. Kawaoka, "A system for functional analysis of Ebola virus glycoprotein.", Proc. Natl. Acad. Sci. USA , vol. 94, pp. 14764-14769 (1997) Wool-Lewis, RJ and P. Bates, "Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines.", J. Virol., Vol. 72, pp. 3155-3160 (1998) Sanchez, A., SG Trappier, BW Mahy, CJ Peters, and ST Nichol, "The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing.", Proc. Natl. Acad. Sci. USA, vol. 93, pp. 3602-3607 (1996) Volchkov, VE, S. Becker, VA Volchkova, VA Ternovoj, AN Kotov, SV Netesov, and H.-D. Klenk, "GP mRNA of Ebola virus is edited by the Ebola virus polymerase and by T7 and vaccinia virus polymerases." , Virology, vol. 214, pp. 421-430 (1995) Feldmann, H. and MP Kiley, "Classification, structure, and replication of filoviruses.", Curr. Top. Microbiol. Immunol., Vol. 235, pp. 1-22 (1999) Alvarez, CP, F. Lasala, J. Carrillo, O. Muniz, AL Corbi, and R. Delgado, "C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans.", J. Virol., Vol. 76, pp. 6841-6844 (2002) Baribaud, F., S. Pohlmann, and RW Doms, "The role of DC-SIGN and DC-SIGNR in HIV and SIV attachment, infection, and transmission.", Virology, vol. 286, pp. 1-6 (2001 ) Baribaud, F., S. Pohlmann, G. Leslie, F. Mortari, and RW Doms, "Quantitative expression and virus transmission analysis of DC-SIGN on monocyte-derived dendritic cells.", J. Virol., Vol. 76, pp. 9135-9142 (2002) Simmons, G., JD Reeves, CC Grogan, LH Vandenberghe, F. Baribaud, JC Whitbeck, E. Burke, MJ Buchmeier, EJ Soilleux, LJ Riley, RW Doms, P. Bates, and S. Pohlmann, "DC-SIGN and DC-SIGNR bind ebola gycoproteins and enhance infection of macrophages and endothelial cells. ", Virology, vol. 305, pp. 115-123 (2003) Lin, G., G. Simmons, S. Pohlmann, F. Baribaud, H. Ni, GJ Leslie, BS Haggarty, P. Bates, D. Weissman, JA Hoxie, and RW Doms, "Differential N-linked glycosylation of Human Immunodeficiency Virus and Ebola Virus envelope glycoproteins modulates interactions with DC-SIGN and DC-SIGNR. ", J. Virol., Vol. 77, pp. 1337-1346 (2003) Oda S., Sato M., Toyoshima S. and Osawa T., "Purification and characterization of a lectin-like molecule specific for galactose / N-acetylgalactosamine from tumoricidal macrophages.", J. Biochem. (Tokyo), vol. 104 , pp. 600-605 (1988) Suzuki N, Yamamoto K, Osawa T, Irimura T., "Molecular cloning and expression of cDNA encoding human macrophage C-type lectin: its unique carbohydrate specificity for Tn antigen.", J. of Immunology, vol. 156: pp. 128 -135 (1996) Tsuiji M., Fujimori M., Ohashi Y., Higashi N., Onami TM, Hedrick, SM, Irimura T., "Molecular cloning and characterization of a novel mouse macrophage C-type lectin, mMGL2, which has a distinct carbohydrate specificity from mMGL1. ", J. Biol. Chem., vol. 277, pp. 28892-28901 (2002) Denda-Nagai, K., Kubota N., Tsuiji M., Kamata M. and Irimura T., "Macrophage C-type lectin on bone marrow-derived immature dendritic cells is involved in the initialization of glycosilated antigens.", Glycobiology, vol. 12, pp. 443-450 (2002) Higashi, N., Morikawa A., Fujioka K., Fujita Y., Sano Y., Miyata-Takeuchi M., Suzuki N. and Irimura T., "Human macrophage lectin specific for galactose / N-acetylgalactosamine is a marker for cells at an intermediate stage in their differentiation from monocytes into macrophages. ", Int. Immunol., vol. 14, pp. 545-554 (2002) Higashi, N., K. Fujioka, K. Denda-Nagai, S. Hashimoto, S. Nagai, T. Sato, Y. Fujita, A. Morikawa, M. Tsuiji, M. Miyata-Takeuchi, Y. Sano, N Suzuki, K. Yamamoto, K. Matsushima, and T. Irimura, "The macrophage C-type lectin specific for galactose / N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells.", J. Boil. Chem ., vol. 277, pp. 20686-20693. (2002)

  An object of the present invention is to provide a method for preventing and / or treating a viral infection, particularly an infection caused by a virus having a mucin-type glycoprotein typified by filovirus, and a pharmaceutical composition therefor.

  In addition to DC-SIGN and / or L-SIGN, the involvement of MGL in the viral infection process was revealed. Furthermore, it has been clarified that the infection can be remarkably suppressed by inhibiting the interaction between a specific sugar chain on the virus particle and a cell surface lectin molecule specific for the sugar chain. It was.

  Accordingly, a first aspect of the present invention is a method for preventing and / or treating an infection caused by a virus having a mucin-type (O-linked) sugar chain, which method is used in a patient in need of such treatment. Inhibiting the interaction between macrophage galactose-type C-type lectin (MGL) on macrophages and mucin-type sugar chains on viral particles is provided.

  The method preferably comprises administering to a patient in need of such treatment an effective amount of a specific binding partner for MGL, such as an anti-MGL antibody or a reactive fragment of the antibody. Alternatively, the method can include administering to a patient in need of such treatment an effective amount of solubilized MGL protein, eg, a protein comprising the extracellular domain of hMGL.

  Further, in filovirus, DC-SIGN and L-SIGN are also involved in the virus infection. Therefore, in the present invention, DC-SIGN and / or L-SIGN and the virus on MIR are simultaneously detected. It is also preferable to inhibit the sugar chain interaction. Suitable means for inhibiting such interactions include simultaneously or sequentially administering to a patient in need of such treatment an effective amount of a specific binding partner for MGL or solubilized MGL protein. It may be exemplified by administering a specific binding partner for DC-SIGN and / or L-SIGN or solubilized DC-SIGN and / or L-SIGN protein.

  Examples of viruses having a mucin-type sugar chain to which the method of the present invention is suitable are filoviruses, that is, Ebola and Marburg virus, and the method of the present invention is particularly effective in these viral infections. .

  Another aspect of the present invention provides a pharmaceutical composition for preventing and / or treating an infection caused by a virus having the mucin-type (O-linked) sugar chain. Preferably, the pharmaceutical composition comprises a therapeutically effective amount of a specific binding partner for macrophage galactose type C lectin (MGL), together with a pharmaceutically acceptable carrier.

  The present invention is characterized by controlling infection by the virus by inhibiting the interaction between MGL and mucin-type sugar chains on virus particles, typically mucin-type sugar chains on envelope GP. To do.

  Here, the mucin-type sugar chain (also referred to as O-linked sugar chain) generally refers to a sugar chain in which the N-acetylgalactosamine is O-glycosidically bonded to the hydroxyl group of serine or threonine. . It is widely known that proteins present in virus particles can be modified by such sugar chains. Therefore, the term “virus having a mucin-type (O-linked type) sugar chain” used in the present invention includes Virus particles represented by envelope GP can include viruses modified with the mucin-type sugar chain. Examples of the virus include, but are not limited to, viruses of the family Filoviridae, that is, Ebola virus and Marburg virus.

  That is, the present invention is based on the elucidation of the significant involvement of MGL in the process of establishing the infection of the “virus having a mucin-type (O-linked sugar chain)”, thereby preventing the function of the lectin. Enables the treatment and prevention of the viral infection in mammals. Further, in the present invention, it has been demonstrated that the use of a specific binding partner for MGL, for example, an anti-MGL antibody, is effective in blocking such MGL function.

  The anti-MGL antibody that can be preferably used in the present invention may be prepared by any method known to those skilled in the art using whole MGL or a partial peptide thereof as an antigen, and includes polyclonal antibodies and monoclonal antibodies. It belongs to any of the five classes (IgG, IgA, IgM, IgD, IgE) classified as immunoglobulin structures, physicochemical properties and immunological properties, or subclasses depending on the type of heavy chain. Also good. Typically, when the antibody of the present invention is obtained in the form of a polyclonal antibody, the polyclonal antibody is prepared from the antiserum of an animal immunized using MGL or a partial sequence thereof (such as a synthetic peptide) as an immunogen. Specifically, for the antiserum from the immunized animal, for example, the immunogen containing an adjuvant is subcutaneously injected into the immunized animal, and the subcutaneous administration is performed a predetermined number of times (for example, 5 times) at an appropriate interval (for example, 1 week). It can be obtained by repeatedly collecting whole blood after final immunization and separating it. Such a method is described in, for example, CURRENT PROTOCOLS IN IMMUNOLOGY, Chapter 2.4 (Publisher: John Wiley & Sons, Inc., New York). Subsequently, the polyclonal antibody is purified from the antiserum by using MGL used for animal immunization or a partial peptide thereof as a chromatography resin, such as CNBr activated Sepharose or HiTrap NHS-activated (both manufactured by Amersham Pharmacia). It is immobilized by covalent bonding, and the antiserum is applied to the immobilized resin to specifically adsorb the antibody in the antiserum on the resin, and then the antibody adsorbed on the resin is adsorbed to an appropriate buffer or Although it can also be achieved by elution and recovery using chaotropic ions or the like, it is not limited to this.

  When the antibody of the present invention is obtained as a monoclonal antibody, spleen cells and myeloma cells of laboratory animals immunized with MGL, preferably rodents such as mice, rats and hamsters, using techniques known to those skilled in the art. A parent cell for cell fusion such as a cell line is fused, a suitable one is selected from the obtained hybridomas, cloned, and then the fused cells are cultured in vitro or in vivo, and more specific than this culture mixture. Collect highly monoclonal antibodies.

  hMGL cDNA is described in Suzuki N, Yamamoto K, Osawa T, Irimura T., "Molecular cloning and expression of cDNA encoding human macrophage C-type lectin: its unique carbohydrate specificity for Tn antigen.", J. of Immunology, vol. 156: cloned in pp. 128-135 (1996). Therefore, MGL as an immunogen can be prepared by a method known per se to those skilled in the art based on these cDNAs.

  Particularly preferred anti-hMGL antibodies are supra, Suzuki N, Yamamoto K, Osawa T, Irimura T., “Molecular cloning and expression of cDNA encoding human macrophage C-type lectin: its unique carbohydrate specificity, which is incorporated herein by reference. for Tn antigen. ", J. of Immunology, vol. 156: pp. 128-135 (1996), or Higashi, N., Morikawa A., Fujioka K., Fujita Y., Sano Y., Miyata-Takeuchi M ., Suzuki N. and Irimura T., "Human macrophage lectin specific for galactose / N-acetylgalactosamine is a marker for cells at an intermediate stage in their differentiation from monocytes into macrophages.", Int. Immunol., Vol.14, pp It can be prepared based on the description of 545-554 (2002).

In the present invention, a reactive fragment of the antibody obtained by enzymatic digestion of the above antibody may be used. Examples of such antibody fragments include Fab fragments, Fab ′ fragments, F (ab ′) ₂ fragments, F (v) fragments, H chain monomers or dimers, L chain monomers or dimers, one H chain and one Examples include dimers composed of L chains. The fragment can be obtained, for example, by digesting a complete antibody with a protease such as pepsin or papain, or by treating with a reducing agent as necessary after digestion. The H chain and L chain monomers can also be obtained by treating the complete antibody with a reducing agent such as dithiothreitol and then separating the purified chain. Accordingly, in the present invention, the term “specific binding partner” includes these reactive fragments of antibodies, or fusion proteins of the fragments and other proteins.

  In the present invention, in order to inhibit the interaction between MGL and mucin-type sugar chains as described above, it is considered that one of the preferred embodiments is to administer solubilized MGL protein. The solubilization of the MGL can also be achieved by, for example, intact hMGL by appropriate chemical modification, for example, by introducing a polyethylene glycol side chain, and such protein chemical modification itself is known to those skilled in the art. More preferably, the extracellular domain of hMGL can be expressed in a suitable host using a recombinant technique, recovered and purified, and used as the solubilized MGL protein of the present invention. For example, Suzuki N, Yamamoto K, Osawa T, Irimura T., "Molecular cloning and expression of cDNA encoding human macrophage C-type lectin: its unique carbohydrate specificity for Tn antigen.", J of Immunology, vol. 156: pp. 128-135 (1996), recombinant hMGL from cDNA encoding the putative extracellular domain of hMGL can be suitably used for this purpose. In addition, fusion proteins between the extracellular domain and other proteins could be used.

  In the present invention, it is more preferable to inhibit the interaction between DC-SIGN and / or L-SIGN and its ligand in parallel with the inhibition of the interaction between MGL and mucin-type glycoprotein. Specific methods of such functional inhibition of DC-SIGN or L-SIGN are described in International Publication Nos. WO 00/63251 and International Publication No. WO 01/64752, which are incorporated herein by reference. The anti-DC-SIGN antibody or solubilized DC-SIGN protein described in these international publications is a particularly preferred example applicable in the present invention.

  When the antibody of the present invention (including a reactive fragment thereof) or a solubilized protein is used for pharmaceutical purposes, a pharmacologically acceptable method of use having a purity suitable for use as a pharmaceutical product Is preferably used.

  The antibody and solubilized protein of the present invention may be used by directly dissolving or suspending them in a suitable solvent, and if necessary, a pharmaceutically acceptable auxiliary ingredient may be added to the tablet. Or a solid preparation such as capsule, granule, powder or suppository; or a liquid preparation such as syrup, elixir or injection, and these can be prepared by a usual method in the pharmaceutical field. In the case of a liquid preparation, it may be dissolved or suspended in water or other appropriate medium at the time of use. In particular, in the case of injections, they may be dissolved or suspended in physiological saline or glucose solution as necessary, and buffering agents and preservatives may be added. Accordingly, pharmacologically acceptable auxiliary ingredients include solvents, bases, stabilizers, preservatives, solubilizers, excipients, buffers, etc., such as gelatin, lactose, sucrose, titanium oxide, Starch, crystalline cellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, corn starch, microcrystalline wax, white petrolatum, magnesium aluminate metasilicate, anhydrous calcium phosphate, citric acid, trisodium citrate, hydroxypropylcellulose, sorbitol, sorbitan fatty acid ester, polysorbate, Sucrose fatty acid ester, polyoxyethylene hydrogenated castor oil, polyvinyl pyrrolidone, magnesium stearate, light anhydrous silicic acid, talc, vegetable oil, benzyl alcohol, gum arabic, propylene glycol, polyal Glycols, although cyclodextrin or hydroxypropyl cyclodextrin and the like, but is not limited thereto.

  When the antibody and solubilized protein of the present invention are in the above-mentioned dosage form, the administration method and dosage are set according to the age, sex, disease type, and degree of the patient. be able to. That is, those therapeutically effective amounts are administered orally, or by inhalation, transdermal administration, eye drops, intravaginal administration, intraarticular administration, rectal administration, intravenous administration, local administration, intramuscular administration, subcutaneous administration, intraperitoneal administration. An appropriate method may be selected and administered from the above, and the composition for such administration may contain 1 to 100% by weight, preferably 10 to 80% by weight of the antibody of the present invention. Can be contained.

  The dose and frequency of administration of the antibody and solubilized protein, etc. in the above composition vary depending on the gender, age, weight, symptom of the patient, and the type and range of the intended treatment, but are generally administered orally. In this case, 1 to 50 mg / kg per day for an adult is divided into 1 to several times. In the case of parenteral administration, 1 to 10 mg / kg is preferably divided into 1 to several times.

  Without further explanation, those skilled in the art given the above explanation can make full use of the present invention. The following examples are given for illustrative purposes only.

Interaction between mucin-type sugar chains on Ebola virus and MGL plays an important role in Ebola virus infection process
I. Method (1) Virus Ebola virus: Mayinga strain of Zaire species was used.

  Vesicular stomatitis virus (VSV): Serotype Indiana was used.

VSVΔG ^* -ZaireGP: against Ebola virus (Zaire strain) against a virus strain (VSVΔG ^* ) that lacks the vesicular stomatitis virus G protein (VSV G protein) and is further modified to express GFP (Green Fluorescent Protein) Envelope glycoprotein (GP) was introduced (Takada, A., C. Robison, H. Goto, A. Sanchez, KG Murti, MA Whitt, and Y. Kawaoka, "A system for functional analysis of Ebola virus glycoprotein." , Proc. Natl. Acad. Sci. USA, vol. 94, pp. 14764-14769 (1997)).

VSVΔG ^* -ZaireGPΔmuc: a pseudovirus in which Ebola virus GP (ZaireGPΔmuc) lacking a mucin-like domain (amino acids 311-466) is introduced in place of the intact Zaire strain envelope glycoprotein (ZaireGP) in the above VSVΔG ^* -ZaireGP (Simmons, G., RJ Wool-Lewis, F. Baribaud, RC Netter, and P. Bate, "Ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence.", J. Virol., vol. 76, pp. 2518-2528 (2002)).

VSVΔG ^* -VSVG: As a control, VSVΔG ^* -VSVG, which is a strain in which VSV G protein was introduced into the above VSVΔG ^* , was used (see Takada (1997) above).

  Each virus was grown in Vero E6 cells and stored at −80 ° C. until use.

(2) Cells Monkey kidney Vero E6 cells and human chronic myeloid leukemia cells (K562) were cultured in DMEM medium and RPMI 1640 medium supplemented with 10% fetal calf serum, L-glutamine and antibiotics, respectively.

  All infectious substances, including Ebola virus, were handled in a Biosafety Level 4 facility at the Canadian Science Center of Human and Animal Health.

(3) Expression of human MGL As a human MGL gene, the coding sequence of hMGL6A (Suzuki N, Yamamoto K, Osawa T, Irimura T., “Molecular cloning and expression of cDNA encoding human macrophage C-type lectin: its unique carbohydrate specificity for Tn antigen. ", J. of Immunology, vol. 156: pp. 128-135 (1996) and Higashi, N., K. Fujioka, K. Denda-Nagai, S. Hashimoto, S. Nagai, T. Sato, Y. Fujita, A. Morikawa, M. Tsuiji, M. Miyata-Takeuchi, Y. Sano, N. Suzuki, K. Yamamoto, K. Matsushima, and T. Irimura, "The macrophage C-type lectin specific for galactose / N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells. ", J. Boil. Chem., Vol. 277, pp. 20686-20693. (2002)), pRc, an expression vector for mammalian cells. / CMV (manufactured by Invitrogen) was inserted in the sense direction (CR4) and the antisense direction (CR7: control). K562 cells were transformed with the above plasmid by electroporation, and transformants were selected with Geneticin (G418 sulfate, manufactured by GIBCO). In addition, hMGL6A ⁺ cells can be obtained from Higashi, N., Morikawa A., Fujioka K., Fujita Y., Sano Y., Miyata-Takeuchi M., Suzuki N. and Irimura T., “Human macrophage lectin specific for galactose / N-acetylgalactosamine is a marker for cells at an intermediate stage in their differentiation from monocytes into macrophages. ", Int. Immunol., Vol. 14, pp. 545-554 (2002). ) Were enriched using immunomagnetic beads prepared using Transformants were then cloned by limiting dilution and CR4-4 or CR4-10 as MGL expressing cell clones and CR7 clones as controls were used in subsequent infection experiments.

(4) In the experiments using viral infection experiments pseudotyped virus for transformants, 10 ⁵ cells / clones K562 transformants (including CR7 clones as a control) were introduced Ebola virus GP VSV ( Pseudotype Ebola virus: VSVΔG ^* -ZaireGP and VSVΔG ^* -ZaireGPΔmuc), or VSV pseudotype virus (VSVΔG ^* -VSVG; control). The infection rate was evaluated by measuring the number of GFP positive K562 cells by FACS and standardizing the result for CR7 clone as 100.

In an infection experiment on K562 cells with true Ebola virus and VSV, the TCID ₅₀ of the virus was previously measured in Vero cells, and each K562 transformant (10 ⁷ cells / clone) had a TCID ₅₀ (of Vero cells) of 10 ⁵ . Cells were infected in proportions. Thereafter, the supernatant of the cell cultures on the 1st to 3rd days was collected, and the infectious titer of virus (TCID ₅₀ / ml) per unit volume of the supernatant was measured as TCID ₅₀ in Vero cells.

II. Results II-1) Pseudotype Ebola virus infection experiment The results of the infection experiment are shown in FIG. VSVΔG ^* -ZaireGP pseudotype Ebola virus (hereinafter abbreviated as “ZaireGP”) that retains a mucin-like domain is a transformant that expresses hMGL, CR4-4 and CR4- It was revealed that 10 clones showed a very high infection rate. Further, from the results of infection experiments with VSVΔG ^* -VSVG (hereinafter referred to as “VSVG”), which is a pseudotype virus of VSV, the infection rate of VSVG to hMGL transformants CR4-4 and CR4-10 was found to be CR7 of the control. It was concluded that the increase in viral infection in MGL expressing cells was specific for Ebola virus, since it was comparable to that for clones.

It has also been shown that deletion of a mucin-like domain from Ebola virus GP significantly reduces the infectivity of the virus to hMGL expressing cells. That is, in the pseudo type VSVΔG ^* -ZaireGPΔmuc lacking a mucin-like domain (hereinafter abbreviated as “ZaireGPΔmuc”), only a slight increase in the infection rate in hMGL-expressing cells against CR7 was observed. Did not show a significant increase in the infection rate observed with ZaireGP.

  The above results show that in addition to the previous findings regarding the role of DC-SIGN and L-SIGN in fibrovirus infections including Ebola, MGL, which is a lectin having a specificity different from these, has shown a significant impact on the virus infection. In particular, the role of the interaction between mucin-type sugar chains on viruses and MGL on cells was demonstrated.

II-2) Infection experiment with true Ebola virus and VSV The results of measuring the virus infectivity titer (TCID ₅₀ / ml) in the cell culture supernatant on days 1, 2 and 3 after cell infection with virus are shown in FIG. Shown in HMGL expression in K562 cells does not affect the infection of the cells to VSV-virus particle production. However, in Ebola virus, the production of the virus particles by hMGL-expressing cells reaches 100 times or more. In particular, in the control CR7, infectious Ebola virus particles were not produced in the supernatant at the multiplicity of infection used in this experiment. This result also clearly supports the involvement of hMGL in Ebola virus infection.

Inhibition of the interaction between mucin-type glycans and MGL on Ebola virus can control Ebola virus infection process
I. Method (1) Virus VSVΔG ^* -ZaireGP and VSVΔG ^* -VSVG (control) of Example 1 were used.

(2) Cells As described in Example 1.

(3) Expression of human MGL As described in Example 1.

(4) Antibody MLD-1: MLD-1, which is an anti-human MGL monoclonal antibody, is produced by Higashi, N., Morikawa A., Fujioka K., Fujita Y., Sano Y., Miyata-Takeuchi M., Suzuki N. and Irimura T., "Human macrophage lectin specific for galactose / N-acetylgalactosamine is a marker for cells at an intermediate stage in their differentiation from monocytes into macrophages.", Int. Immunol., vol. 14, pp. 545-554 (2002).

(5) Virus infection inhibition experiment by antibody Cells (10 ⁵ cells / clone) of each clone were incubated with the MLD-1 (as 200-fold diluted ascites fluid) or normal mouse ascites fluid (control) for 30 minutes. Subsequently, ZaireGP or VSV G was infected in the presence of the antibody. The method for evaluating the infection rate is the same as in Example 1 for pseudotyped viruses.

II. Results The results are shown in FIG. In ZaireGP infection, administration of anti-hMGL antibody significantly reduces the infection rate of the virus. From the results of VSVG, it can be seen that this effect is extremely remarkable in viruses having mucin type (O-linked type) sugar chains represented by Ebola virus, and inhibition of the interaction between mucin type sugar chains on MGL-virus. Has been shown to be effective in preventing and / or treating infection with viruses having the sugar chain.

  In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing and / or treating an infection caused by a virus having a mucin-type (O-linked) sugar chain. Preferably, the pharmaceutical composition comprises a therapeutically effective amount of a specific binding partner or solubilized MGL protein for macrophage galactose type C lectin (MGL), together with a pharmaceutically acceptable carrier.

FIG. 1 shows VSVΔG ^* -ZaireGP (ZaireGP), a pseudotype virus of Ebola virus prepared based on vesicular stomatitis virus (VSV), and VSVΔG ^* in which the mucin-like domain is deleted in the virus GP ^. -ZaireGPΔmuc (ZaireGPΔmuc) and VSVΔG ^* -VSVG (VSVG) as controls were infected with hMGL expressing transformants (CR4-4 and CR4-10) and control (CR7) of human chronic myeloid leukemia cells (K562). The result of having measured the infection rate at the time of FACS by FACS is shown. In the figure, the infected cells are measured by FACS, and the background infection rate is subtracted from each data. In addition, the number of infected cells of CR7 as a control is normalized to 100, and the relative infection rate (%) is expressed. The experiment was performed in triplicate and shows representative data. FIG. 2 shows the infection rates of hMGL-expressing transformants (CR4-10) and control (CR7) by authentic VSV (control) and true Ebola virus (Ebola) on days 1, 2, and 3 after infection. Expressed as virus infectivity titer (TCID ₅₀ ) in culture supernatant (1 ml). The experiment was performed in duplicate and representative data were shown. FIG. 3 shows the effect of anti-human MGL antibody on viral infection. The effect of anti-hMGL monoclonal antibody MLD-1 (hMGL Ab), which is an anti-human MGL antibody, on pseudotyped Ebola virus (ZaireGP) and VSV (VSV G) infection, and expression of hMGL in human chronic myeloid leukemia cells (K562) Comparison is made in the transformant (CR4-10) and the control (CR7). A control antibody (Control Ab) was added as normal mouse ascites fluid. Representative data were shown in triplicate experiments. The number of infected cells of CR7 as a control was normalized to 100, and the relative infection rate (%) was expressed.

Claims (15)

  A method for preventing and / or treating an infection caused by a virus having a mucin-type (O-linked) sugar chain, wherein the method comprises a macrophage galactose-type C lectin (on macrophages) in a patient in need of such treatment. The method comprising inhibiting the interaction between MGL) and a mucin-type sugar chain on a virus particle.   2. The method of claim 1, wherein the inhibition of the interaction comprises administering to a patient in need of such treatment an effective amount of a specific binding partner for MGL or solubilized MGL protein.   3. The method of claim 2, wherein the specific binding partner is an anti-MGL antibody or a reactive fragment of the antibody.   The method of claim 2, wherein the solubilized MGL protein comprises the extracellular domain of hMGL.   Furthermore, the method as described in any one of Claims 1 thru | or 4 which inhibits interaction between DC-SIGN and / or L-SIGN, and the N-linked sugar chain on a virus particle.   The method according to any one of claims 1 to 5, wherein the virus having a mucin-type (O-linked type) sugar chain is a virus of the family Filoviridae.   The method according to claim 6, wherein the Filoviridae virus is an Ebola virus.   A pharmaceutical composition for the prevention and / or treatment of an infection caused by a virus having a mucin-type (O-linked) sugar chain, the composition against a therapeutically effective amount of macrophage galactose type C lectin (MGL) Said pharmaceutical composition characterized in that it comprises a specific binding partner or solubilized MGL protein.   9. The pharmaceutical composition according to claim 8, wherein the specific binding partner is an anti-MGL antibody or a reactive fragment of the antibody.   9. The pharmaceutical composition according to claim 8, wherein the solubilized MGL protein comprises the extracellular domain of hMGL.   The pharmaceutical composition according to any one of claims 8 to 10, further comprising a specific binding partner for DC-SIGN and / or L-SIGN or a solubilized DC-SIGN and / or L-SIGN protein.   The specific binding partner for DC-SIGN is an anti-DC-SIGN antibody or a reactive fragment of the antibody, and the specific binding partner for L-SIGN is an anti-L-SIGN antibody or a reactive fragment of the antibody. The pharmaceutical composition according to claim 11.   12. The pharmaceutical composition according to claim 11, wherein the solubilized DC-SIGN and / or L-SIGN protein comprises the extracellular domain of those proteins.   The pharmaceutical composition according to any one of claims 8 to 13, wherein the virus having a mucin-type (O-linked) sugar chain is a virus of the family Filoviridae.   15. The pharmaceutical composition according to claim 14, wherein the Filoviridae virus is an Ebola virus.

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