JP7729583B2 - Preventive or therapeutic drug for inflammatory bowel disease - Google Patents

Description

The present invention relates to a drug for the prevention or treatment of inflammatory bowel disease.

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 to form, 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).

As mentioned above, the cause of inflammatory bowel disease is unknown, so conventional diarrhea medications are ineffective. Aminosalicylate preparations (sulfasalazine, 5-aminosalicylic acid) and corticosteroid preparations have traditionally been widely used as first- and second-line medications for the treatment of inflammatory bowel disease. In severe cases, immunosuppressants (azathioprine, 6-mercaptopurine, etc.) and anticytokine preparations are also used. While sulfasalazine and 5-aminosalicylic acid are widely used aminosalicylate preparations, 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 granulocytopenia, hemolytic anemia, and folate deficiency anemia. Corticosteroid preparations 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 from these conventional therapies, and uses 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 also in maintaining remission. Known side effects include hypertension, chills, rash, fever, headache, and eczema. In addition, infections requiring antibiotics and carcinogenicity are considered 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, refractory cases exist, making the development of new therapeutic agents an urgent issue.

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 ORAIIP). This secreted form of eIF5A is a protein in which the tyrosine residues of eIF5A are sulfated, and they have found 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 caused by oxidative stress and suppress myocardial ischemia-reperfusion injury (Patent Document 1, Non-Patent Document 1).

International Publication No. 2009/144933

SCIENTIFIC REPORTS 5, 13737 (2015)

The objective of the present invention is to provide a new preventive or therapeutic agent for inflammatory bowel disease, for which the development of an effective therapeutic agent is desired.

As a result of investigations aimed at developing a therapeutic agent for the aforementioned difficult-to-treat diseases, the inventors discovered that neutralizing antibodies against secretory eIF5A exhibit excellent preventive and therapeutic effects against inflammatory bowel disease, leading to the completion of the present invention.

That is, the present invention provides the following [1] to [16].

[1] A preventive or therapeutic drug for inflammatory bowel disease, which contains as an active ingredient a neutralizing antibody against secretory eIF5A.
[2] The preventive or therapeutic agent according to [1], wherein the neutralizing antibody against secretory 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, 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, 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, wherein one to several amino acids are 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 secretory 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 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: 1 with one to several 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: 2 or the amino acid sequence shown in SEQ ID NO: 2 with one to several amino acids deleted, substituted or added; a heavy chain CDR3 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 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: 4 with one to several 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 to several 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 to several 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 secretory 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 against secreted eIF5A according to [9] or [10], wherein the neutralizing antibody 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, 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, wherein one to several amino acids are deleted, substituted or added; a light chain CDR2 which is a polypeptide consisting of 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, 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, which comprises administering an effective amount of a neutralizing antibody against secretory eIF5A.
[14] The method described in [13], wherein the neutralizing antibody against secreted eIF5A is a monoclonal antibody.
[15] The method of [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: 1 with one to several 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: 2 or the amino acid sequence shown in SEQ ID NO: 2 with one to several amino acids deleted, substituted or added; a heavy chain CDR3 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 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: 4 with one to several 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 to several 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 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.

This invention makes it possible to prevent or treat inflammatory bowel disease, a designated intractable disease for which no effective therapeutic agents have been available until now.

FIG. 1 shows the effect of a neutralizing antibody against secreted eIF5A on weight loss in inflammatory bowel disease model animals. FIG. 1 shows the effect of a neutralizing antibody against secreted eIF5A on the reduction in colon length in inflammatory bowel disease model animals. FIG. 1 shows the effect of a neutralizing antibody against secreted eIF5A on disease activity (DAI) in an animal model of inflammatory bowel disease. 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. 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. FIG. 1 shows the expression of ORAIP in colonic mucosal cells of inflammatory bowel disease model animals. The amino acid sequence of the heavy chain variable region is shown. The amino acid sequence of the light chain variable region is shown.

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

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 (deoxyhypusine eIF5A) by deoxyhypusine synthase (DHS), then hypusinated (hypusinated eIF5A) by deoxyhypusine hydroxylase (DOHH). This hypusinated eIF5A is known to exhibit cell proliferation-inducing properties. 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 strongly 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 is unknown.

The neutralizing antibody used in the present invention may be of any origin, type (monoclonal or polyclonal), or form, as long as it binds to the secreted eIF5A protein. Specifically, known antibodies such as mouse antibodies, rat antibodies, avian antibodies, human antibodies, chimeric antibodies, 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 under NITE P-02955.

Preferred examples of neutralizing antibodies used in the present invention include neutralizing antibodies 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: 1 with one to several 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: 2 or the amino acid sequence shown in SEQ ID NO: 2 with one to several amino acids deleted, substituted or added; a heavy chain CDR3 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 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: 4 with one to several 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 to several 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 to several 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,
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 and the original amino acid sequence is preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more.

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 the 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.

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 a conventional immunization method, followed by fusing the resulting immune cells with known parent cells by a conventional cell fusion method, and screening for monoclonal antibody-producing cells by a conventional screening method.
Specifically, monoclonal antibodies can be prepared as follows.

Purified secreted eIF5A protein, eIF5A protein, and partial peptides of eIF5A protein containing tyrosine residues that can be sulfated can be used as sensitizing antigens. In this case, partial peptides can be obtained by chemical synthesis from 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. There are no limitations on the portion or size of the human eIF5A protein used as the partial peptide.

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. Rodents, such as mice, rats, and hamsters, as well as birds, rabbits, and monkeys, are generally used.

Animals are immunized with a sensitizing antigen according to known methods. For example, a common method involves injecting the sensitizing antigen intraperitoneally or subcutaneously 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. An appropriate carrier can also be used during immunization with the sensitizing antigen. In particular, when using a partial peptide with a small molecular weight as the sensitizing antigen, it is desirable to conjugate it to a carrier protein such as albumin or keyhole limpet hemocyanin before immunization.

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

Mammalian myeloma cells are used as the other parent cell to be fused with the immune cells. These myeloma cells can be any of a variety of 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. al., Nature (1978) 276, 269-270), F.O. al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I.S.J. Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133), etc. are preferably used.

Cell fusion between the immune cells and myeloma cells can basically be carried out 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).

More specifically, the cell fusion is carried out in a standard nutrient culture medium in the presence of a cell fusion promoter. Examples of cell fusion promoters that can be used include polyethylene glycol (PEG) and Sendai virus (HVJ). If desired, an adjuvant such as dimethyl sulfoxide can also be added to enhance fusion efficiency.

The ratio of immune cells to myeloma cells can be set as desired. For example, a ratio of immune cells to myeloma cells of 1 to 10 is preferred. The culture medium used for the cell fusion can be, for example, RPMI 1640 culture medium, MEM culture medium, or other standard culture medium used for this type of cell culture, which are suitable for growing the myeloma cell line. Furthermore, serum supplements such as fetal calf serum (FCS) can also be used in combination.

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

The hybridomas obtained in this manner 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 period of time (usually several days to several weeks) sufficient for cells other than the desired hybridoma (non-fused cells) to die. Then, a conventional limiting dilution method is performed to screen for and single-clone hybridomas that produce the desired antibody.

Screening and single cloning of the desired antibody can be performed using a screening method based on known antigen-antibody reactions. 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 using limiting dilution or other methods. In this case, the antigen used for immunization can be used as the antigen.

The hybridomas producing monoclonal antibodies produced in this way can be subcultured in standard culture medium and can be stored for long periods in liquid nitrogen.

To obtain monoclonal antibodies from the hybridoma, the hybridoma is cultured according to conventional methods and the culture supernatant is obtained, or the hybridoma is administered to a compatible mammal for proliferation and the ascites fluid is obtained. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

The neutralizing antibodies used in the present invention are not limited to whole antibody molecules, but may be antibody fragments or modified forms 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')2, Fv, Fab/c, which has one Fab and an intact Fc, and single-chain Fv (scFv), in which the Fv of the heavy or light 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, M.S. 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.). Press, Inc. , Lamoyi, E. , Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al. , Methods in Enzymology (1989) 121, 663-669, Bird, R. E. et al. , TIBTECH (1991) 9, 132-137).

An scFv can be obtained by linking the heavy chain V region and light chain V region of an antibody. In this scFv, the heavy chain V region and light chain V region are linked via a linker, preferably a peptide linker (Huston, J.S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The heavy chain V region and light 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.

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

In addition, 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 standard methods, and scFv can be obtained using the host according to standard methods.

Fragments of these antibodies 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 fragments of these antibodies.

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, etc. Bispecific antibodies can be produced by combining the HL pairs of two types of antibodies, or they 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 using genetic engineering techniques.

As will be shown in the Examples below, neutralizing antibodies against secretory eIF5A have excellent preventive and therapeutic effects on 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 for inflammatory bowel disease 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.).

The pharmaceutical of the present invention can be formulated and used in the form of a pharmaceutical composition by mixing, dissolving, granulating, tableting, emulsifying, encapsulating, lyophilizing, or otherwise combining the neutralizing antibody with a pharmaceutically acceptable carrier well known in the art.

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, along with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc.

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.

The dosage and frequency of administration will vary depending on the dosage form and route of administration, as well as the patient's symptoms, age, and body weight. Generally, neutralizing antibodies can be administered once or several times a day, or once every one to two weeks, at a dose ranging from about 0.001 mg to 1000 mg per kg of body weight per day, preferably from about 0.01 mg to 10 mg.

The present invention will now be described in detail using examples, but the present invention is not limited to these examples.

Example 1
(Preparation of neutralizing antibodies against secreted eIF5A)
The anti-ORAIP antibody (clone YSP5-45-36) was prepared using a peptide consisting of amino acid residues (44-72) of human eIF5A protein (including the lysine residue at position 50 to be hypusinated and the tyrosine residue at position 69 to be 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 Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE) under accession number P-02955.

Example 2
(Analysis of CDR regions of monoclonal antibodies)
Total cytoplasmic RNA was recovered from the hybridoma clone YSP5-45-36.
cDNA was synthesized by RT reaction using primers specific to the mouse IgG (H chain, L chain) sequence.
Using the synthesized cDNA as a template, 5' RACE analysis was carried out using SMARTer ^™ RACE 5'/3' Kit (TaKaRa Code Z4859N).
The resulting consensus sequence was analyzed for CDR regions using the analysis tool IMGT ^™ , the international ImMunoGeneTics information system ^™ 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.

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, ORAIIP mAb) 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. On day 0 and days 8 to 11, the mice were given distilled water to drink.
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 is thought to have been reduced 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. This experimental design included a recovery period after the end of DSS administration, but it is estimated that the difference would have been even greater if sacrifice had been performed at the end of DSS and colon length measured. From the above, it is thought that anti-ORAIP antibodies are effective against ulcerative colitis.

(2) Figure 3 shows the results of an analysis of the effect of anti-ORAIP antibody administration on ulcerative colitis disease activity using the DAI (Disease Activity Index) 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 administration group compared to the IgG administration control group.

(3) Figures 4 and 5 show HE staining and TUNEL staining (and methyl green nuclear staining) of colon wall tissue 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). 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, both 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). As described above, it was demonstrated that anti-ORAIP antibodies significantly inhibit DSS-induced destruction of mucosal epithelial cells (apoptosis induction).

Next, Figure 6 shows the results of a fluorescent staining analysis of ORAIIP expression in colonic wall tissue from the normal control group (Normal) and the DSS-IgG group. While ORAIIP expression was barely observed in the epithelial cells on the mucosal surface of the normal control group (upper row, arrow), significant ORAIIP expression was observed in a large number of what appeared to be densely packed epithelial cells in the DSS-IgG group (lower row, arrow and enlarged view). This suggests that ulcerative colitis induced by DSS loading may induce ORAIIP expression primarily in mucosal epithelial cells, thereby inducing apoptosis.

Claims (2)

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