Preventive or therapeutic drug for inflammatory bowel disease

Abstract

To provide a novel prophylactic or therapeutic agent for inflammatory bowel disease, for which development of an effective therapeutic agent is desired.SOLUTION: Provided is a prophylactic or therapeutic agent for inflammatory bowel disease, the agent comprising a neutralizing antibody against secretory eIF5A as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a preventive or therapeutic agent for inflammatory bowel disease. [Background technology]

[0002] Inflammatory bowel disease (IBD) is an incurable disease of unknown cause that causes chronic inflammation or ulcers in the mucosa of the large and small intestine, resulting in long-term diarrhea, bloody stools, and repeated recurrences. There are two main types of IBD: Crohn's disease (CD) and ulcerative colitis (UC). Crohn's disease, also known as regional enteritis, granulomatous ileitis, or ileocolitis, is a chronic inflammation of the intestinal wall that can occur in any part of the digestive tract. Ulcerative colitis is a chronic disease in which inflammation of the large intestine causes ulcers, resulting in attacks accompanied by bloody diarrhea, severe abdominal pain, and fever. While the number of patients with both diseases is not as high as in Europe or the United States, they are steadily increasing in Japan, with approximately 220,000 patients with ulcerative colitis (2020) and approximately 70,000 patients with Crohn's disease (2020).

[0003] As mentioned above, the cause of inflammatory bowel disease is unknown, so conventional antidiarrheal medications are ineffective. Aminosalicylates (sulfasalazine, 5-aminosalicylic acid) and corticosteroids have traditionally been widely used as first- and second-line treatments for inflammatory bowel disease. In severe cases, immunosuppressants (e.g., azathioprine, 6-mercaptopurine) and anticytokine agents have also been used. While sulfasalazine and 5-aminosalicylic acid are widely used aminosalicylates, approximately 50% of patients who receive them develop gastrointestinal disorders such as nausea, vomiting, loss of appetite, and liver dysfunction, as well as blood disorders such as agranulocytopenia, hemolytic anemia, and folate deficiency anemia. Corticosteroids are associated with various side effects, including osteoporosis, growth retardation, secondary adrenal insufficiency, impaired glucose tolerance, and hypertension. On the other hand, anti-cytokine therapy is a completely different treatment approach from these conventional approaches. It utilizes the chimeric anti-human TNF-α monoclonal antibody infliximab and the human anti-human TNF-α monoclonal antibody adalimumab. It has been reported to be effective in patients with steroid-resistant, moderate-to-severe Crohn's disease and to maintain remission. Known side effects include hypertension, chills, rash, fever, headache, and eczema. Furthermore, infections requiring antibiotics and carcinogenicity are concerns. In ulcerative colitis, the involvement of oxidative stress-mediated apoptosis in the cell damage mechanism has been suggested, but this mechanism remains unclear. Currently, treatments such as aminosalicylic acid preparations, steroids, and anti-cytokine therapy are used, but severe cases remain refractory, making the development of new therapeutic agents an urgent issue.

[0004] Meanwhile, the present inventors have investigated components secreted extracellularly under hypoxia-reoxygen conditions, i.e., under oxidative stress conditions, and discovered a secreted form of eIF5A (named ORAIP). They found that this secreted form of eIF5A is a protein in which tyrosine residues of eIF5A are sulfated, and that it induces apoptosis in cells subjected to oxidative stress. Furthermore, they have found that neutralizing antibodies against this secreted form of eIF5A inhibit apoptosis induced by oxidative stress and suppress myocardial ischemia-reperfusion injury (Patent Document 1, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2009 / 144933 Brochure [Non-patent literature]

[0006] [Non-Patent Document 1] SCIENTIFIC REPORTS 5,13737(2015) Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a novel prophylactic or therapeutic agent for inflammatory bowel diseases, for which the development of an effective therapeutic agent is desired. [Means for solving the problem]

[0008] The present inventors conducted research to develop therapeutic agents for the above-mentioned difficult-to-treat diseases, and as a result discovered that neutralizing antibodies against secreted eIF5A exhibit excellent preventive and therapeutic effects against inflammatory bowel diseases, thereby completing the present invention.

[0009] That is, the present invention provides the following [1] to

[16] .

[0010] [1] A preventive or therapeutic drug for inflammatory bowel disease, which contains as an active ingredient a neutralizing antibody against secreted eIF5A. [2] The preventive or therapeutic agent according to [1], wherein the neutralizing antibody against secreted eIF5A is a monoclonal antibody. [3] The prophylactic or therapeutic agent according to [1] or [2], wherein the neutralizing antibody against secreted eIF5A has a heavy chain CDR1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 3, with one to several amino acids deleted, substituted or added; a light chain CDR1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, or the amino acid sequence shown in SEQ ID NO: 5, or the amino acid sequence shown in SEQ ID NO: 6, with one to several amino acids deleted, substituted or added. [4] The preventive or therapeutic drug according to any one of [1] to [3], wherein the inflammatory bowel disease is ulcerative colitis. [5] Use of a neutralizing antibody against secreted eIF5A for the manufacture of a drug for the prevention or treatment of inflammatory bowel disease. [6] The use according to [5], wherein the neutralizing antibody against secreted eIF5A is a monoclonal antibody. [7] The use according to [5] or [6], wherein the neutralizing antibody against secreted eIF5A is a neutralizing antibody having a heavy chain CDR1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 with one or more amino acids deleted, substituted or added; a heavy chain CDR2 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3 with one or more amino acids deleted, substituted or added; a light chain CDR1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4 with one or more amino acids deleted, substituted or added; a light chain CDR2 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5 with one or more amino acids deleted, substituted or added; and a light chain CDR3 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6 with one or more amino acids deleted, substituted or added. [8] The use according to any one of [5] to [7], wherein the inflammatory bowel disease is ulcerative colitis. [9] A neutralizing antibody against secreted eIF5A for use in the prevention or treatment of inflammatory bowel disease.

[10] The neutralizing antibody according to [9], wherein the neutralizing antibody against secreted eIF5A is a monoclonal antibody.

[11] The neutralizing antibody according to [9] or

[10] , wherein the neutralizing antibody against secreted eIF5A has a heavy chain CDR1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 3, wherein one to several amino acids are deleted, substituted or added; a heavy chain CDR2 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 3, wherein one to several amino acids are deleted, substituted or added; a light chain CDR1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, or the amino acid sequence shown in SEQ ID NO: 5, or the amino acid sequence shown in SEQ ID NO: 5, wherein one to several amino acids are deleted, substituted or added; and a light chain CDR3 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or the amino acid sequence shown in SEQ ID NO: 6, wherein one to several amino acids are deleted, substituted or added.

[12] The neutralizing antibody according to any one of [9] to

[11] , wherein the inflammatory bowel disease is ulcerative colitis.

[13] A method for preventing or treating inflammatory bowel disease, comprising administering an effective amount of a neutralizing antibody against secreted eIF5A.

[14] The method described in

[13] , wherein the neutralizing antibody against secreted eIF5A is a monoclonal antibody.

[15] The method described in

[13] or

[14] , wherein the neutralizing antibody against secreted eIF5A is a neutralizing antibody having a heavy chain CDR1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 3, with one to several amino acids deleted, substituted or added; a light chain CDR1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 5, or the amino acid sequence shown in SEQ ID NO: 6, with one to several amino acids deleted, substituted or added.

[16] The method according to any one of

[13] to

[15] , wherein the inflammatory bowel disease is ulcerative colitis. [Effects of the Invention]

[0011] According to the present invention, it becomes possible to prevent or treat inflammatory bowel disease, which is designated as an intractable disease and for which no effective therapeutic agent has been available until now. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the effect of a neutralizing antibody against secreted eIF5A on weight loss in inflammatory bowel disease model animals. [Figure 2] FIG. 1 shows the effect of a neutralizing antibody against secreted eIF5A on the reduction in colon length in inflammatory bowel disease model animals. [Figure 3] FIG. 1 shows the effect of a neutralizing antibody against secreted eIF5A on disease activity (DAI) in inflammatory bowel disease model animals. [Figure 4] FIG. 1 shows the effect of a neutralizing antibody against secreted eIF5A on colonic mucosal tissue damage (HE staining) in inflammatory bowel disease model animals. [Figure 5] FIG. 1 shows the effect of a neutralizing antibody against secreted eIF5A on apoptosis (TUNEL staining) of colonic mucosal cells in inflammatory bowel disease model animals. [Figure 6] FIG. 1 shows the expression of ORAIP in colonic mucosal cells of inflammatory bowel disease model animals. [Figure 7] The amino acid sequence of the heavy chain variable region is shown. [Figure 8] The amino acid sequence of the light chain variable region is shown. DETAILED DESCRIPTION OF THE INVENTION

[0013] The active ingredient of the pharmaceutical of the present invention is a neutralizing antibody against secreted eIF5A.

[0014] Eukaryotic translation initiation factor (eIF) 5A, as its name suggests, has been identified as a translation initiation factor. eIF5A is expressed in the cytoplasm and is deoxyhypusinated by deoxyhypusine synthase (DHS) (deoxyhypusine eIF5A), which is then hypusinated by deoxyhypusine hydroxylase (DOHH) (hypusinated eIF5A). This hypusinated eIF5A is known to exert cell proliferation-inducing effects. Furthermore, eIF5A is secreted extracellularly and converted to secreted eIF5A, in which tyrosine residues are sulfated. This secreted eIF5A induces apoptosis in cells subjected to oxidative stress. Furthermore, neutralizing antibodies against this secreted eIF5A potently suppress apoptosis induced by oxidative stress and protect against myocardial and cerebral ischemia-reperfusion injury (Patent Document 1, Non-Patent Document 1). However, the effect of this neutralizing antibody on inflammatory bowel disease remains unknown.

[0015] The neutralizing antibody used in the present invention may be any antibody capable of binding to secreted eIF5A protein, regardless of its origin, type (monoclonal or polyclonal), or form. Specifically, known antibodies such as mouse, rat, avian, human, chimeric, and humanized antibodies can be used. While the antibody may be a polyclonal antibody, a monoclonal antibody is preferred. The monoclonal antibody is preferably a monoclonal antibody produced by a hybridoma deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE P-02955).

[0016] Preferred examples of neutralizing antibodies used in the present invention include neutralizing antibodies having a heavy chain CDR1 that is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 with one or more amino acids deleted, substituted, or added; a heavy chain CDR2 that is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 with one or more amino acids deleted, substituted, or added; a heavy chain CDR3 that is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3 with one or more amino acids deleted, substituted, or added; a light chain CDR1 that is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4 with one or more amino acids deleted, substituted, or added; a light chain CDR2 that is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5 with one or more amino acids deleted, substituted, or added; and a light chain CDR3 that is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6 with one or more amino acids deleted, substituted, or added. Further preferred examples of neutralizing antibodies include those having a heavy chain variable region that is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence in which one to several amino acids have been deleted, substituted, or added, and a light chain variable region that is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence in which one to several amino acids have been deleted, substituted, or added, in the amino acid sequence shown in SEQ ID NO: 8. Here, the amino acid sequence in which one or several amino acids have been deleted, substituted, or added is preferably an amino acid sequence in which one to four amino acids have been deleted, substituted, or added, and more preferably an amino acid sequence in which one to three amino acids have been deleted, substituted, or added. The identity of the amino acid sequence in which one or several amino acids have been deleted, substituted, or added to the original amino acid sequence is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more.

[0017] The neutralizing antibodies used in the present invention can be obtained as polyclonal or monoclonal antibodies using known methods. Mammalian- or avian-derived monoclonal antibodies are preferred as neutralizing antibodies used in the present invention. Mammalian-derived monoclonal antibodies are particularly preferred. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced in hosts transformed by genetic engineering techniques with expression vectors containing antibody genes.

[0018] Monoclonal antibody-producing hybridomas can basically be prepared using known techniques as follows: eIF5A protein in which tyrosine residues are sulfated, eIF5A protein, hypusinated eIF5A protein in which tyrosine residues are sulfated, hypusinated eIF5A protein, or partial peptides of these proteins, etc., are used as sensitizing antigens, and immunization is carried out using these antigens according to conventional immunization methods. The resulting immune cells are fused with known parent cells by conventional cell fusion techniques, and monoclonal antibody-producing cells are screened by conventional screening methods. Specifically, monoclonal antibodies can be prepared as follows.

[0019] Purified secreted eIF5A protein, eIF5A protein, or a partial peptide of eIF5A protein containing a tyrosine residue that can be sulfated can be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis based on the amino acid sequence of human eIF5A protein, by incorporating a portion of the eIF5A gene into an expression vector, or by degrading natural human eIF5A protein with a protease. The portion and size of the human eIF5A protein used as the partial peptide are not limited.

[0020] The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected taking into consideration compatibility with the parent cells used in cell fusion. Generally, rodents such as mice, rats, and hamsters, or birds, rabbits, and monkeys are used.

[0021] Immunization of animals with a sensitizing antigen is carried out according to known methods. For example, a common method involves intraperitoneal or subcutaneous injection of the sensitizing antigen into a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount of PBS (Phosphate-Buffered Saline) or physiological saline, and if desired, mixed with an appropriate amount of a conventional adjuvant, such as Freund's complete adjuvant. After emulsification, the resulting mixture is administered to the mammal several times every 4 to 21 days. A suitable carrier can also be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it is desirable to conjugate it to a carrier protein such as albumin or keyhole limpet hemocyanin before immunization.

[0022] After immunizing a mammal in this manner and confirming that the desired antibody level is elevated in the serum, immune cells are collected from the mammal and subjected to cell fusion, with preferred immune cells being spleen cells in particular.

[0023] The other parent cell to be fused with the immune cell is a mammalian myeloma cell, which may be any of various known cell lines, such as P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler, G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies, D.H. et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S.F. et al., Nature (1978) 276, 269-270), or FO (de St. Groth, S.F. et al., Nature (1978) 276, 269-270). al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, ISJ Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133), etc. are preferably used.

[0024] The cell fusion between the immune cells and myeloma cells can be carried out basically according to known methods, such as the method of Kohler and Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

[0025] More specifically, the cell fusion is carried out in a conventional nutrient medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), and if desired, an adjuvant such as dimethyl sulfoxide can be added to enhance the fusion efficiency.

[0026] The ratio of immune cells to myeloma cells can be set as desired. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. The culture medium used for the cell fusion may be, for example, RPMI1640 culture medium, MEM culture medium, or other conventional culture medium used for culturing cells of this type, which are suitable for growing the myeloma cell line. Furthermore, serum supplements such as fetal calf serum (FCS) may also be used in combination.

[0027] Cell fusion is performed by thoroughly mixing a predetermined amount of the immune cells and myeloma cells in the culture medium, adding a PEG solution (e.g., an average molecular weight of approximately 1000-6000) preheated to approximately 37°C, usually at a concentration of 30-60% (w / v), and mixing to form the desired fused cells (hybridomas). Subsequently, an appropriate culture medium is successively added, and the mixture is centrifuged to remove the supernatant, thereby repeatedly removing cell fusion promoters and other substances that are undesirable for hybridoma growth.

[0028] The hybridomas thus obtained are selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT culture medium is continued for a sufficient time (usually several days to several weeks) to allow cells other than the target hybridoma (non-fused cells) to die. Then, a conventional limiting dilution method is performed to screen for and single-clone hybridomas that produce the target antibody.

[0029] Screening and single cloning of the desired antibody can be performed using a screening method based on a known antigen-antibody reaction. For example, an antigen can be bound to a carrier, such as polystyrene beads or a commercially available 96-well microtiter plate, and reacted with the hybridoma culture supernatant. After washing the carrier, an enzyme-labeled secondary antibody or the like can be reacted to determine whether the culture supernatant contains the desired antibody that reacts with the sensitizing antigen. Hybridomas producing the desired antibody can be cloned by limiting dilution or other methods. In this case, the antigen used for immunization can be used as the antigen.

[0030] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a normal culture medium and can be stored for a long period in liquid nitrogen.

[0031] Monoclonal antibodies can be obtained from the hybridoma by culturing the hybridoma according to a conventional method and obtaining the culture supernatant, or by administering the hybridoma to a compatible mammal to grow it and obtaining the monoclonal antibody as ascites. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

[0032] The neutralizing antibodies used in the present invention are not limited to whole antibody molecules, but may be antibody fragments or modified fragments thereof, including both bivalent and monovalent antibodies, as long as they bind to and neutralize secreted eIF5A protein. Examples of antibody fragments include Fab, F(ab'), Fv, Fab / c having one Fab and an intact Fc, and single-chain Fv (scFv) in which the Fv of the H chain or L chain is linked via an appropriate linker. Specifically, antibodies are treated with enzymes such as papain or pepsin to generate antibody fragments, or genes encoding these antibody fragments are constructed and introduced into expression vectors, which are then expressed in appropriate host cells (see, for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, A.H. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in See Enzymology (1989) 121, 663-669; Bird, RE et al., TIBTECH (1991) 9, 132-137).

[0033] An scFv can be obtained by linking the H chain V region and L chain V region of an antibody. In this scFv, the H chain V region and L chain V region are linked via a linker, preferably a peptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). The H chain V region and L chain V region in the scFv may be derived from any of the antibodies described herein. The peptide linker linking the V regions can be, for example, any single-chain peptide consisting of 12 to 19 amino acid residues.

[0034] DNA encoding scFv can be obtained by using the DNA encoding the H chain or H chain V region of the antibody and the DNA encoding the L chain or L chain V region, respectively, where the entire sequence or a DNA portion encoding a desired amino acid sequence is used as a template and amplifying it by PCR using a primer pair that specifies both ends of the DNA, and then further amplifying it using a primer pair that specifies DNA encoding a peptide linker portion so that both ends of the DNA are linked to the H chain and L chain, respectively.

[0035] Furthermore, once DNA encoding scFv has been prepared, an expression vector containing it and a host transformed with the expression vector can be obtained according to conventional methods, and by using the host, scFv can be obtained according to conventional methods.

[0036] These antibody fragments can be produced in a host by obtaining and expressing the genes in the same manner as described above. The "antibody" of the present invention also includes these antibody fragments.

[0037] Furthermore, the neutralizing antibody used in the present invention may be a bispecific antibody. A bispecific antibody may have antigen-binding sites that recognize different epitopes on a molecule, or one antigen-binding site may recognize the secreted IF5A protein and the other antigen-binding site may recognize a labeling substance or the like. Bispecific antibodies can be produced by combining the HL pairs of two types of antibodies, or can be obtained by fusing hybridomas that produce different monoclonal antibodies to produce bispecific antibody-producing fusion cells. Furthermore, bispecific antibodies can also be produced by genetic engineering techniques.

[0038] As will be shown in the Examples below, neutralizing antibodies against secreted eIF5A have excellent preventive and therapeutic effects against inflammatory bowel diseases. Inflammatory bowel diseases include ulcerative colitis and Crohn's disease, and the neutralizing antibodies of the present invention are useful for the prevention and treatment of ulcerative colitis. In particular, with regard to ulcerative colitis, the neutralizing antibodies of the present invention specifically exhibit the effect of significantly suppressing weight loss and reduction in colon length. Furthermore, because the neutralizing antibody of the present invention has a different mechanism of action from conventional therapeutic agents for inflammatory bowel disease, particularly ulcerative colitis, it can be used in combination with such conventional therapeutic agents for ulcerative colitis. Examples of other therapeutic agents that can be used in combination include aminosalicylic acid preparations (sulfasalazine, 5-aminosalicylic acid), corticosteroid preparations, immunosuppressants (azathioprine, 6-mercaptopurine, etc.), and anti-cytokine preparations (anti-TNF-α antibodies, etc.).

[0039] The medicament of the present invention can be used in the form of a pharmaceutical composition prepared by mixing, dissolving, granulating, tableting, emulsifying, encapsulating, lyophilizing, or the like the neutralizing antibody together with a pharmaceutically acceptable carrier well known in the art.

[0040] For oral administration, the neutralizing antibody can be formulated into dosage forms such as tablets, pills, sugar-coated tablets, soft capsules, hard capsules, solutions, suspensions, emulsions, gels, syrups, and slurries together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, and the like.

[0041] For parenteral administration, the neutralizing antibody can be formulated into dosage forms such as injectable solutions, suspensions, emulsions, creams, ointments, inhalants, and suppositories, along with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, and the like. For injection formulations, the neutralizing antibody can be dissolved in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. Furthermore, the composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, the pharmaceutical composition can be prepared in powder form, and an aqueous solution or suspension can be prepared using sterile water or the like before use. For inhalation administration, the neutralizing antibody can be powdered and mixed with a suitable base such as lactose or starch to form a powder mixture. Suppositories can be prepared by mixing the neutralizing antibody with a conventional suppository base such as cocoa butter. Furthermore, the pharmaceutical of the present invention can be encapsulated in a polymer matrix or the like to form a sustained-release formulation.

[0042] The dosage and frequency of administration will vary depending on the dosage form and route of administration, as well as the symptoms, age, and body weight of the patient. In general, the neutralizing antibody can be administered once or several times a day, or once every one to two weeks, to a range of approximately 0.001 mg to 1000 mg, preferably approximately 0.01 mg to 10 mg, per kg of body weight per day. [Example]

[0043] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

[0044] Example 1 (Generation of neutralizing antibodies against secreted eIF5A) The anti-ORAIP antibody (clone YSP5-45-36) was produced using a peptide consisting of amino acid residues (44-72) of human eIF5A protein (including lysine residue 50, which is hypusinated, and tyrosine residue 69, which is sulfated) conjugated to keyhole limpet hemocyanin (KLH) as the antigen. A hybridoma was generated by cell fusion of splenocytes from mice immunized with this antigen with mouse myeloma cells. From the monoclonal antibodies produced by the hybridoma, an antibody specifically reacting with amino acid residues (44-72) of the human eIF5A protein was selected by ELISA, and a monoclonal antibody (clone YSP5-45-36) was established. The resulting hybridoma was deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary under NITE P-02955.

[0045] Example 2 (Analysis of CDR regions of monoclonal antibodies) Total cytoplasmic RNA was recovered from the hybridoma producing clone YSP5-45-36. cDNA was synthesized by RT reaction using primers specific to mouse IgG (H chain, L chain) sequences. Using the synthesized cDNA as a template, SMARTer TM 5' RACE analysis was performed using the RACE 5' / 3' Kit (TaKaRa Code Z4859N). The resulting consensus sequence was analyzed using the IMGT analysis tool. TM , the international ImMunoGeneTics information system TM Analysis of the CDR regions was performed using http: / / www.imgt.org. Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and CDR3 are shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. The heavy chain variable region is shown in SEQ ID NO: 7 and Figure 3, and the light chain variable region is shown in SEQ ID NO: 8 and Figure 4.

[0046] Example 3 (Effective against ulcerative colitis) (1) Five-week-old C57BL / 6J male mice (five mice per group) were intraperitoneally injected with the neutralizing antibody of the present invention (anti-ORAIP antibody, ORAIPmAb) or mouse IgG (control) (day 0), and then administered dextran sodium sulfate (DSS) mixed with drinking water every day from day 1 to day 7. Distilled water was administered on day 0 and days 8 to 11. From day 5 after administration of dextran sulfate sodium, the mice developed diarrhea and bloody stools, and exhibited symptoms of ulcerative colitis. In contrast, these symptoms were reduced in the group administered the neutralizing antibody of the present invention. Changes in body weight over time were measured (Figure 1), and colon length was measured on day 10 (Figure 2). Although the difference with the control group was likely smaller due to a slight recovery from the peak of inflammation on day 8, the colon length was significantly longer in the anti-ORAIP antibody group. Although the experimental design included a recovery period after the end of DSS administration, it is likely that the difference would have been even greater if colon length had been measured after sacrifice at the end of DSS. These findings suggest that anti-ORAIP antibodies are effective against ulcerative colitis.

[0047] (2) Figure 3 shows the results of an analysis of the effect of anti-ORAIP antibody administration on ulcerative colitis disease activity using the Disease Activity Index (DAI) score. The DAI score is a numerical sum of weight loss, stool consistency, and the degree of blood in the stool, using the following indices. It is an established index used in multiple publications. At the peak on day 8, the DAI score was significantly suppressed in the anti-ORAIP antibody-administered group compared to the IgG-administered control group.

[0048] [Table 1]

[0049] (3) HE and TUNEL staining (and methyl green nuclear staining) of colonic wall tissues from the normal control group (Normal), the group administered mouse IgG followed by DSS challenge (DSS-IgG group), and the group administered anti-ORAIP antibody followed by DSS challenge (DSS-anti-ORAIP antibody group) are shown in Figures 4 and 5, respectively. HE staining revealed that the ductal structure of the villi was maintained and the epithelial cells were aligned in the normal control group, whereas the ductal structure was absent and the epithelial cell alignment was completely disrupted in the DSS-IgG group. In contrast, the ductal structure and epithelial cells were partially maintained in the DSS-anti-ORAIP antibody group. Furthermore, TUNEL staining revealed that most cells in the normal control group were negative, whereas many epithelial cells in the DSS-IgG group underwent apoptosis (arrows). In contrast, apoptosis was observed in only a small number of cells in the DSS-anti-ORAIP antibody group (arrows). These results demonstrate that anti-ORAIP antibody significantly inhibits DSS-induced destruction of mucosal epithelial cells (apoptosis induction).

[0050] Next, the expression of ORAIP in colonic wall tissues from the normal control group (Normal) and the DSS-IgG group was analyzed by fluorescent staining. Figure 6 shows the results. While ORAIP expression was barely observed in epithelial cells on the mucosal surface in the normal control group (upper row, arrow), significant ORAIP expression was observed in a large number of densely packed epithelial cells in the DSS-IgG group (lower row, arrow and enlarged view). This suggests that DSS-induced ulcerative colitis may induce apoptosis primarily through the induction of ORAIP expression in mucosal epithelial cells.

Claims

1. A preventive or therapeutic drug for inflammatory bowel disease, comprising a neutralizing antibody against secretory eIF5A as an active ingredient, A preventive or therapeutic drug for inflammatory bowel disease, wherein the neutralizing antibody against secreted eIF5A is a monoclonal antibody having a heavy chain CDR1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR3 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 which is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

6.

2. 2. The preventive or therapeutic drug according to claim 1, wherein the inflammatory bowel disease is ulcerative colitis.

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