JP2021155343A - Agent for preventing and/or treating pulmonary hypertension - Google Patents

Abstract

To provide a novel agent for preventing and/or treating pulmonary hypertension, for which an effective treatment agent is desired to be developed.SOLUTION: An agent for preventing and/or treating pulmonary hypertension has a neutralizing antibody to eIF5A with a sulfated tyrosine residue as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to a prophylactic or therapeutic agent for pulmonary hypertension.

Pulmonary hypertension, unlike normal hypertension, has a poor prognosis that causes heart and lung dysfunction due to increased blood pressure (pulmonary arterial pressure) in the pulmonary blood vessels, which are the blood vessels that send blood from the heart to the lungs. It is a group of progressive diseases. Specifically, it is a disease in which the average pulmonary artery pressure in the resting supine position is 25 mmHg or more by performing a right heart catheter examination. These pulmonary hypertension are pulmonary arterial hypertension (Group 1), pulmonary hypertension associated with left ventricular disease (Group 2), pulmonary hypertension associated with respiratory disease or hypoxia (Group 3), chronic thrombosis and / or embolism. It is classified into pulmonary hypertension due to a disease (Group4) and pulmonary hypertension of unknown cause (Group5).

As drug therapy for these pulmonary hypertension, prostacyclin derivatives, endothelin receptor antagonists, phosphodiesterase 5 inhibitors, soluble guanylate cyclase stimulants and the like are used as pulmonary vasodilators. Among them, (idiopathic and hereditary) pulmonary arterial hypertension is a drug-resistant and poor prognosis, and is an intractable rare disease with less than 3,000 patients in Japan.

On the other hand, the present inventor investigated the components secreted extracellularly under hypoxic-reoxygen conditions, that is, under oxidative stress conditions, and discovered a secretory eIF5A (named ORAIP). It was found that the secretory eIF5A is a protein in which the tyrosine residue of eIF5A is sulfated and induces apoptosis of cells subjected to oxidative stress. Furthermore, it has been found that a neutralizing antibody against the secretory eIF5A (ORAIP) suppresses apoptosis due to oxidative stress and suppresses myocardial / cerebral ischemia-reperfusion injury (Patent Document 1, Non-Patent Documents 1 and 2). ..

International Publication No. 2009/144933 Pamphlet

SCIENTFIC REPORTS 5, 13737 (2015) SCIENTFIC REPORTS 9, 13512 (2019)

An object of the present invention is to provide a new preventive or therapeutic agent for pulmonary hypertension for which the development of an effective therapeutic agent is desired.

As a result of studies for developing a therapeutic agent for pulmonary hypertension, which is difficult to treat, the present inventor locally highly expresses secretory eIF5A (ORAIP) protein in the lung tissue of a pulmonary hypertension model rat. When a neutralizing antibody against the secretory eIF5A (ORAIP) was administered, a significant decrease in pulmonary arterial pressure, a significant decrease in right ventricular systolic pressure, and pulmonary arteries in pulmonary tissue were observed. The present invention has been completed by finding that the neutralizing antibody exhibits an excellent prophylactic and therapeutic effect on pulmonary hypertension.

That is, the present invention provides the following [1] to [12].

[1] A prophylactic or therapeutic agent for pulmonary hypertension, which comprises a neutralizing antibody against secretory eIF5A (ORAIP) as an active ingredient.
[2] The prophylactic or therapeutic agent according to [1], wherein the neutralizing antibody against the secretory eIF5A (ORAIP) is a monoclonal antibody.
[3] The neutralizing antibody against secretory eIF5A (ORAIP) is a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and an amino acid represented by SEQ ID NO: 3. Heavy chain CDR3 containing the sequence, light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence set forth in SEQ ID NO: 6. The prophylactic or therapeutic agent according to [1] or [2], which is a neutralizing antibody having.
[4] Use of neutralizing antibody against secretory eIF5A (ORAIP) for the production of prophylactic or therapeutic agents for pulmonary hypertension.
[5] The use according to [4], wherein the neutralizing antibody against the secretory eIF5A (ORAIP) is a monoclonal antibody.
[6] The neutralizing antibody against secretory eIF5A (ORAIP) is a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and an amino acid shown in SEQ ID NO: 3. Heavy chain CDR3 containing the sequence, light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence set forth in SEQ ID NO: 6. Use according to [4] or [5], which is a neutralizing antibody having.
[7] A neutralizing antibody against secretory eIF5A (ORAIP) for use in the prevention or treatment of pulmonary hypertension.
[8] The neutralizing antibody according to [7], wherein the neutralizing antibody against the secretory eIF5A (ORAIP) is a monoclonal antibody.
[9] The neutralizing antibody against secretory eIF5A (ORAIP) is a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and an amino acid represented by SEQ ID NO: 3. Heavy chain CDR3 containing the sequence, light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence set forth in SEQ ID NO: 6. The neutralizing antibody according to [7] or [8], which is a neutralizing antibody having.
[10] A method for preventing or treating pulmonary hypertension, which comprises administering an effective amount of a neutralizing antibody against eIF5A in which a tyrosine residue is sulfated.
[11] The method according to [10], wherein the neutralizing antibody against the secretory eIF5A (ORAIP) is a monoclonal antibody.
[12] The neutralizing antibody against secretory eIF5A (ORAIP) is a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and an amino acid represented by SEQ ID NO: 3. Heavy chain CDR3 containing the sequence, light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence set forth in SEQ ID NO: 6. The method according to [10] or [11], which is a neutralizing antibody having.

When the medicament or the pharmaceutical composition of the present invention is used, an excellent therapeutic effect on pulmonary hypertension can be obtained. Pulmonary hypertension is a disease for which there is no excellent therapeutic agent, and the medicament of the present invention is extremely useful. In addition, the medicament of the present invention includes pulmonary arterial hypertension (Group 1), respiratory disease or pulmonary hypertension associated with hypoxia (Group 3), which mainly consists of pulmonary vascular disorders, among pulmonary hypertension. It is useful for pulmonary hypertension (Group 5) of unknown cause, and is particularly useful for pulmonary arterial hypertension (Group 1), which is designated as an intractable disease.

The experimental method of the drug (monochromotalin) -induced pulmonary hypertension model rat used in this invention is shown. Monocrotalin-induced pulmonary hypertension model-The expression of ORAIP (secretory eIF5A) in the lung tissue of rats is shown. Effect of anti-ORAIP (secretory eIF5A) antibody on monochromatic pulmonary hypertension model rats (A) Pulmonary artery flow deceleration (PAD) and (B) Estimated pulmonary artery pressure (mean PAP; mPAP) The result of the analysis is shown in. The results of analysis of the effect of anti-ORAIP (secretory eIF5A) antibody on monochromatic pulmonary hypertension model rats by right ventricular systolic pressure (RVSP) are shown. The results of analysis of the effect of anti-ORAIP (secretory eIF5A) antibody on monocrotalin-induced pulmonary hypertension model rats by wall thickness of pulmonary arterioles in lung tissue are shown. 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 medicament of the present invention is a neutralizing antibody against secretory eIF5A (ORAIP).

Eukaryotic initiation factor (eIF) 5A is, as the name implies, a substance identified as a translation initiation factor. eIF5A is expressed in the cytoplasm, deoxyhypusine monocytosis (DHS) deoxyhypusine (deoxyhypusine eIF5A), then hypocinylated by deoxyhypusine hydroxylase (DOHH) (hypusine eIF5A), and this hypusin Chemical eIF5A is known to exhibit a cell proliferation effect. In addition, eIF5A is secreted extracellularly in response to oxidative stress stimulation, and changes to secretory eIF5A (ORAIP) in which tyrosine residues are sulfated. The secretory eIF5A (ORAIP) induces apoptosis in oxidatively stressed cells. The neutralizing antibody against this secretory eIF5A (ORAIP) strongly suppresses apoptosis due to oxidative stress and suppresses myocardial / cerebral ischemia-reperfusion injury (Patent Document 1 and Non-Patent Document 2). However, it is not known how this neutralizing antibody acts on pulmonary hypertension.

The neutralizing antibody used in the present invention may bind to a secretory eIF5A (ORAIP) protein, regardless of its origin, type (monoclonal, polyclonal) and shape. Specifically, known antibodies such as mouse antibody, rat antibody, tri-antibody, human antibody, chimeric antibody, and humanized antibody can be used. The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody. The monoclonal antibody is preferably a monoclonal antibody produced by a hybridoma deposited as NITE P-02955 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary.

Preferred examples of the neutralizing antibody used in the present invention include a heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence represented by SEQ ID NO: 3. In having a heavy chain CDR3 containing, a light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence set forth in SEQ ID NO: 6. It is a Japanese antibody. A more preferred example of a neutralizing antibody is a neutralizing antibody having a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 8. Here, the above-mentioned "including an amino acid sequence" includes a case where one to several amino acids are deleted, substituted or added in the amino acid sequence.

The neutralizing antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody by using known means. As the neutralizing antibody used in the present invention, a mammalian-derived or avian-derived monoclonal antibody is preferable. In particular, mammalian-derived monoclonal antibodies are preferred. Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by hosts transformed with an expression vector containing an antibody gene by genetic engineering techniques.

The monoclonal antibody-producing hybridoma can be produced basically by using a known technique as follows. That is, an eIF5A protein in which a tyrosine residue is sulfated, an eIF5A protein, a hypecinated eIF5A protein in which a tyrosine residue is sulfated, a hypecinated eIF5A protein, and a partial peptide of these proteins are used as sensitizing antigens. , This can be produced by immunizing this according to a normal immunization method, fusing the obtained immune cells with a known parent cell by a normal cell fusion method, and screening monoclonal antibody-producing cells by a normal screening method.
Specifically, the following may be used to prepare a monoclonal antibody.

An eIF5A protein in which a purified tyrosine residue is sulfated, an eIFA protein, a partial peptide of an eIF5A protein containing a tyrosine residue that can be sulfated, and the like can be used as a sensitizing antigen. At this time, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of the human eIF5A protein, a part of the eIF5A gene can be incorporated into an expression vector, and a natural human eIF5A protein can be obtained by a proteolytic enzyme. It can also be obtained by disassembling. The portion and size of the human eIF5A protein used as a partial peptide is not limited.

The mammal immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion, and is generally of rodents. Animals such as mice, rats, hamsters, or birds, rabbits, monkeys and the like are used.

To immunize an animal with a sensitizing antigen, a known method is used. For example, as a general method, the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted with PBS (Phosphate-Buffered Saline), physiological saline or the like to an appropriate amount, suspended, and if desired, a normal adjuvant, for example, Freund's complete adjuvant, is mixed in an appropriate amount, and after emulsification, Administer to mammals several times every 4 to 21 days. In addition, a suitable carrier can be used at the time of immunization with a sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, it is desirable to immunize by binding to a carrier protein such as albumin or keyhole limpet hemocyanin.

After immunizing the mammal in this way and confirming that the desired antibody level rises in the serum, immune cells are collected from the mammal and subjected to cell fusion. Examples include cells.

As the other parent cell fused with the immune cell, a mammalian myeloma cell is used. The myeloma cells are derived from various known cell lines such as P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U. 1 (Curent Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6,511-518), MPC-11. (Margulies. DH et al., Cell (1976) 8,405-415), SP2 / 0 (Shumman, M. et al., Nature (1978) 276,269-270), FO (de St. Groth, SF et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trovebridge, ISJ Exp. Med. (1978) 148, 313-323), R210 (Galfle, G. et al., Nature (1979) 277, 131-133) and the like are preferably used.

The cell fusion of the immune cell and the myeloma cell is basically a known method, for example, the method of Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73,3- 46) and the like can be performed.

More specifically, the cell fusion is carried out in a normal nutrient culture medium, for example, in the presence of a cell fusion promoter. As the fusion accelerator, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if desired, an auxiliary agent such as dimethyl sulfoxide can be added and used in order to increase the fusion efficiency.

The usage ratio of immune cells and myeloma cells can be set arbitrarily. For example, it is preferable to increase the number of immune cells to 1 to 10 times that of myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, and other ordinary culture mediums used for this type of cell culture can be used, and further. , A serum supplement such as cow fetal serum (FCS) can also be used in combination.

For cell fusion, a predetermined amount of the immune cells and myeloma cells are well mixed in the culture solution, and a PEG solution (for example, an average molecular weight of about 1000 to 6000) preheated to about 37 ° C. is usually 30 to 60% (for example). By adding at a concentration of w / v) and mixing, the desired fusion cells (hybridoma) are formed. Subsequently, an appropriate culture solution is sequentially added, and the operation of centrifuging to remove the supernatant is repeated to remove a cell fusion agent or the like that is unfavorable for the growth of hybridomas.

The hybridoma thus obtained is selected by culturing in a usual selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). The culture in the above HAT culture medium is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fused cells) to die. The usual limiting dilution method is then performed to screen and single clone hybridomas that produce the antibody of interest.

Screening and single cloning of the target antibody may be performed by a screening method based on a known antigen-antibody reaction. For example, the antigen is bound to a carrier such as beads made of polystyrene or the like or a commercially available 96-well microtiter plate, reacted with the culture supernatant of a hybridoma, the carrier is washed, and then an enzyme-labeled secondary antibody or the like is reacted. Therefore, it can be determined whether or not the culture supernatant contains the antibody of interest that reacts with the sensitizing antigen. A hybridoma that produces the desired antibody can be cloned by a limiting dilution method or the like. At this time, the antigen used for immunization may be used.

The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture medium and can be stored for a long period of time in liquid nitrogen.

To obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant thereof, or the hybridoma is administered to a mammal compatible with the hybridoma to proliferate and used as ascites. The method of obtaining is adopted. The former method is suitable for obtaining high-purity antibody, while the latter method is suitable for mass production of antibody.

The neutralizing antibody used in the present invention is not limited to the whole molecule of the antibody, and may be a fragment of the antibody or a modified product thereof as long as it binds to and neutralizes a secretory eIF5A (ORAIP) protein, and is divalent. Both antibodies and monovalent antibodies are included. For example, as an antibody fragment, Fab, F (ab') 2, Fv, Fab / c having one Fab and a complete Fc, or a single in which H chain or L chain Fv is linked with an appropriate linker. Chain Fv (scFv) can be mentioned. Specifically, the antibody is treated with an enzyme such as papain or pepsin to generate an antibody fragment, or a gene encoding these antibody fragments is constructed and introduced into an expression vector, and then in an appropriate host cell. Express (eg, Co, M.S. et al., J. Immunol. (1994) 152, 2966-2976, Better, M. & Horwitz, A. H. Methods in Enzymeology (1989) 178, 476-496. , Academia Press, Inc., Pepsin, A. & Skera, A. Methods in Antibody (1989) 178, 476-436, Academia Press, Inc., Lamoyi, E. , Rousseaux, J. et al., Methods in Enzymeology (1989) 121, 663-669, Bird, R. E. et al., TIBTECH (1991) 9, 132-137).

scFv is obtained by linking the H chain V region and the L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. US. A. (1988) 85,5879-5883). The H chain V region and the L chain V region in scFv may be derived from any of those described as antibodies herein. As the peptide linker that links the V region, for example, any single-stranded peptide consisting of 12 to 19 amino acid residues is used.

The DNA encoding scFv is a DNA encoding the H chain or the H chain V region of the antibody, and a DNA encoding the L chain or the L chain V region, all of those sequences or a desired amino acid sequence. The encoding DNA portion is used as a template, and amplification is performed by the PCR method using a primer pair that defines both ends thereof, and then the DNA encoding the peptide linker portion and both ends thereof are linked to the H chain and the L chain, respectively. It is obtained by combining and amplifying the primer pairs specified in 1.

Further, once the DNA encoding scFv is prepared, an expression vector containing them and a host transformed by the expression vector can be obtained according to a conventional method, and by using the host, it is usual. ScFv can be obtained according to the method.

Fragments of these antibodies can be obtained, expressed, and produced by the host in the same manner as described above. The "antibody" in the present invention also includes fragments of these antibodies.

Furthermore, the neutralizing antibody used in the present invention may be a bispecific antibody. The bispecific antibody may be a bispecific antibody having an antigen binding site that recognizes different epitopes on the molecule, or one antigen binding site recognizes a secretory eIF5A (ORAIP) protein and the other. The antigen-binding site may recognize a labeling substance or the like. Bispecific antibodies can be prepared by binding HL pairs of two types of antibodies, or by fusing hybridomas that produce different monoclonal antibodies to produce bispecific antibody-producing fused cells. can. Furthermore, it is also possible to produce bispecific antibodies by genetic engineering techniques.

As shown in Examples below, a neutralizing antibody against secretory eIF5A (ORAIP), particularly a neutralizing antibody having a specific amino acid sequence in the CDR region, has an excellent preventive and therapeutic effect on pulmonary hypertension.
Here, with respect to pulmonary hypertension, the neutralizing antibody of the present invention specifically comprises a significant decrease in pulmonary arterial pressure, a significant decrease in right ventricular systolic pressure, and an intrapulmonary pulmonary artery in pulmonary hypertension. Shows significant suppression of wall thickening. Therefore, the neutralizing antibody of the present invention is useful for pulmonary arterial hypertension (Group 1), pulmonary hypertension associated with respiratory disease or hypoxia (Group 3), and pulmonary hypertension of unknown cause (Group 5). In particular, it is useful as a prophylactic and therapeutic agent for pulmonary arterial hypertension (Group1), which is designated as an intractable disease.

The medicament of the present invention is a pharmaceutical product in which the neutralizing antibody is formulated by mixing, dissolving, granulating, tableting, emulsifying, encapsulating, freeze-drying, etc., together with a pharmaceutically acceptable carrier well known in the art. It can be used as a form of the composition.

For oral administration, the neutralizing antibody is used in tablets, pills, sugar coatings, soft capsules, hard capsules, solutions, etc., together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants and the like. , Suspensions, emulsions, gels, syrups, slurries and the like.

For parenteral administration, the neutralizing antibody is used in injection solutions, suspensions, emulsions, creams, etc., together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants and the like. It can be formulated into a dosage form such as an ointment, an inhalant, or a suppository. In injectable formulations, the neutralizing antibody can be dissolved in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer. In addition, the composition can take the form of suspensions, solutions, emulsions, etc. in oily or aqueous vehicles. Alternatively, the pharmaceutical composition may be produced in the form of a powder, and an aqueous solution or suspension may be prepared using sterile water or the like before use. For administration by inhalation, the neutralizing antibody can be powdered into a powder mixture with a suitable base such as lactose or starch. Suppository formulations can be made by mixing neutralizing antibodies with conventional suppository bases such as cocoa butter. Further, the pharmaceutical product of the present invention can be formulated as a continuous release preparation by encapsulating it in a polymer matrix or the like.

The dose 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, but in general, neutralizing antibodies will range from about 0.001 mg to 1000 mg per kg body weight per day, preferably in the range of about 0.001 mg to 1000 mg. It can be administered once to several times daily so that it ranges from about 0.01 mg to 10 mg.

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1
(Preparation of neutralizing antibody against secretory eIF5A (ORAIP))
The anti-ORAIP antibody (clone YSP5-45-36) contains the amino acid residue (hypsinylated 50th lysine residue and sulfated 69th tyrosine residue) of the human eIF5A protein (44-72th). A peptide consisting of (including) bound to keyhole limpet hemocyanin (KLH) was used as an antigen. A hybridoma was created by fusing mouse splenocytes and mouse myeloma cells immunized with this antigen. From the monoclonal antibody produced by the hybridoma, an antibody that specifically reacts with the amino acid residue (44-72th) of the human eIF5A protein described above was selected by the ELISA method, and a monoclonal antibody (clone YSP5-45-36) was established. .. The obtained hybridoma was deposited as NITE P-02955 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary.

Example 2
(Dissolution of CDR regions of monoclonal antibody)
Total cytoplasmic RNA was recovered from clone YSP5-45-36-producing hybridomas.
CDNA was synthesized by RT reaction using primers specific for mouse IgG (H chain, L chain) sequences.
Using the synthesized cDNA as a template, ^{5'RACE cleavage was performed using SMARTer TM} RACE5'/3'Kit (TaKaRa Code Z4859N).
The obtained consensus sequence was used as an analysis tool for IMGT ^TM , the international ImmunoGeneTechs information system ^TM http: // www. imgt. The CDR regions were dissected using 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 FIG. 6, and the light chain variable region is shown in SEQ ID NO: 8 and FIG.

Example 3
(A) FIG. 1 shows an experimental method of a drug (monochromotalin) -induced pulmonary hypertension model rat used in the present invention.
That is, Wister Rat (7-8 weeks old) is intraperitoneally administered with monochrome tallinn (MCT) (60 mg / kg) (Day 0). Anti-ORAIP antibody (or mouse IgG) is administered intravenously (5 mg / kg) on (Day 0) and (Day 14). Evaluate the degree of pulmonary hypertension after 4 weeks (day 28). Evaluation methods include (1) echocardiography: measurement using pulmonary artery blood flow (according to Koskenvuo et al. Int J Cardiovasc Imaging 2010, 26, 509-518), (2) measurement of right ventricular pressure (RVBP), (3) Right ventricular / left ventricular weight ratio (RV / LV) and (4) Thickening of the pulmonary artery were used.

(B) FIG. 2 shows the expression of ORAIP in the lung tissue of a monocotalin-induced pulmonary hypertension model rat. Almost no expression of ORAIP was observed in the lung tissue of normal rats (Day 0; normal, FIG. 2A), whereas clear expression of ORAIP was observed in the lung tissue of pulmonary hypertension (Day 28) (Fig. 2B). , C; arrow).

(C) Fig. 3 shows the effects of anti-ORAIP antibody on monochromatic pulmonary hypertension model rats: (A) pulmonary artery flow deceleration (PAD) and (B) estimated pulmonary artery pressure (mean PAP;). The result of analysis by mPAP) is shown. Both the pulmonary artery flow decay rate (PAD) and the estimated pulmonary artery pressure (mPAP) were significantly lower in the anti-ORAIP antibody-administered group than in the mouse IgG and PBS-administered group.

(D) Fig. 4 shows the results of analysis of the effect of anti-ORAIP antibody on monochromatic pulmonary hypertension model rats by right ventricular systolic pressure (RVSP). Right ventricular pressure measured values of (A) in (1) mouse IgG administration group, (2) anti-ORAIP antibody administration group, (3) phosphate buffer (PBS) administration group, and (4) normal control group. (B) shows the right ventricular systolic pressure of each group. Right ventricular systolic pressure was significantly reduced in the anti-ORAIP antibody-treated group compared to the mouse IgG and PBS-treated group. In addition, the difference (degree of increase) in right ventricular systolic pressure from the normal control group was suppressed to about 50% or less of the mouse IgG-administered group in the anti-ORAIP antibody-administered group (Fig. 4B).

FIG. 5 shows the results of analysis of the effect of the anti-ORAIP antibody on the monorotalin-induced pulmonary hypertension model rat by the wall thickness of the pulmonary arteriole in the lung tissue. (A) shows representative photographs of lung tissues of each group of (1) mouse IgG administration group, (2) 9 anti-ORAIP antibody administration group, and (3) normal control group, which were EVG-stained. As shown by the red arrowhead, wall thickening of the pulmonary arterioles was significantly suppressed in the anti-ORAIP antibody-administered group as compared with the mouse IgG-administered group (Fig. 5B).

Claims (3)

A prophylactic or therapeutic agent for pulmonary hypertension, which comprises a neutralizing antibody against secretory eIF5A (ORAIP) as an active ingredient. The prophylactic or therapeutic agent according to claim 1, wherein the neutralizing antibody against the secretory eIF5A (ORAIP) is a monoclonal antibody. Neutralizing antibody against secretory eIF5A (ORAIP) includes heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and amino acid sequence shown in SEQ ID NO: 3. Neutralization with heavy chain CDR3, light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence set forth in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence set forth in SEQ ID NO: 6. The prophylactic or therapeutic agent according to claim 1 or 2, which is an antibody.

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