WO2020241660A1 - Preventive or therapeutic agent for diabetic retinopathy - Google Patents

Abstract

Provided is a novel preventive or therapeutic agent for diabetic retinopathy against which the development of effective therapeutic agents has been desired.　This preventive or therapeutic agent for diabetic retinopathy contains a neutralizing antibody against secreted eIF5A as an active ingredient.

Description

Preventive or therapeutic agents for diabetic retinopathy

The present invention relates to a prophylactic or therapeutic agent for diabetic retinopathy.

Diabetic retinopathy is a microangiopathy that occurs in the retina due to diabetes, and in addition to causing vision loss due to macular edema, when it worsens, it may lead to blindness with fundus hemorrhage and retinal detachment. To prevent diabetic retinopathy, it is necessary to control the blood glucose level, but when it develops, laser photocoagulation, vitrectomy, and intravitreal steroid or anti-VEGF drug injection therapy are performed. However, the effects of these drug therapies are temporary, and more effective drug therapies are desired.

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 suppresses apoptosis due to oxidative stress and suppresses myocardial ischemia-reperfusion injury (Patent Document 1, Non-Patent Document 1).

International Publication No. 2009/144933 Pamphlet

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

As a result of studies for developing a therapeutic agent for the above-mentioned difficult-to-treat diseases, the present inventor has found that a neutralizing antibody against secretory eIF5A exhibits an excellent prophylactic therapeutic effect on diabetic retinopathy. completed.

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

[1] A prophylactic or therapeutic agent for diabetic retinopathy, which comprises a neutralizing antibody against secretory eIF5A as an active ingredient.
[2] The prophylactic or therapeutic agent according to [1], wherein the neutralizing antibody against the secretory eIF5A is a monoclonal antibody.
[3] The neutralizing antibody against the secretory eIF5A contains 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 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 [1] or [2], which is an antibody.
[4] Use of a neutralizing antibody against secretory eIF5A for the production of a prophylactic or therapeutic drug for diabetic retinopathy.
[5] The use according to [4], wherein the neutralizing antibody against the secretory eIF5A is a monoclonal antibody.
[6] The neutralizing antibody against the secretory eIF5A contains 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 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. Use according to [4] or [5], which is an antibody.
[7] A neutralizing antibody against secretory eIF5A for use in the prevention or treatment of diabetic retinopathy.
[8] The neutralizing antibody according to [7], wherein the neutralizing antibody against the secretory eIF5A is a monoclonal antibody.
[9] The neutralizing antibody against the secretory eIF5A contains 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 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 neutralizing antibody according to [7] or [8], which is an antibody.
[10] A method for preventing or treating diabetic retinopathy, which comprises administering an effective amount of a neutralizing antibody against secretory eIF5A.
[11] The method according to [10], wherein the neutralizing antibody against the secretory eIF5A is a monoclonal antibody.
[12] The neutralizing antibody against the secretory eIF5A contains 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 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 method according to [10] or [11], which is an antibody.

When the drug or pharmaceutical composition of the present invention is used, an excellent therapeutic effect on diabetic retinopathy can be obtained as compared with conventionally used drugs such as anti-VEGF drugs. This disease is a disease for which there is no excellent therapeutic agent, and the medicament of the present invention is extremely useful.

It shows edema and bleeding in a diabetic retinopathy model mouse in which pericytes have disappeared. It shows the edema / bleeding inhibitory effect of VEGF receptor decoy on diabetic retinopathy model mice. The edema / bleeding inhibitory effect of the neutralizing antibody (anti-ORAIP antibody) of the present invention on a diabetic retinopathy model mouse is shown. The edema / bleeding inhibitory effect of the neutralizing antibody (anti-ORAIP antibody) of the present invention on a diabetic retinopathy model mouse is shown. The effects of the anti-VEGF drug and the neutralizing antibody of the present invention (anti-ORAIP antibody) on a pericyte-disappearing diabetic retinopathy model mouse are shown. The effects of the anti-VEGF drug and the neutralizing antibody of the present invention (anti-ORAIP antibody) on a pericyte-disappearing diabetic retinopathy model mouse 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 the secretory eIF5A.

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

The neutralizing antibody used in the present invention may bind to the secretory eIF5A 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. As the monoclonal antibody, a monoclonal antibody produced by a hybridoma deposited as NITE P-02955 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary is preferable.

Preferred examples of the neutralizing antibody used in the present invention include 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 sequence shown in SEQ ID NO: 3. Among 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.

Purified secretory eIF5A protein, eIFA protein, partial peptide of eIF5A protein containing 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 to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, or the like, suspended in an appropriate amount, and if desired, a normal adjuvant, for example, Freund 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 during 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, and the preferred immune cells are particularly splenic. Examples include cells.

Mammalian myeloma cells are used as the other parent cell fused with the immune cells. 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 solution, 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 medium, 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 cell fusion agents and the like that are unfavorable for the growth of hybridomas.

The hybridoma thus obtained is selected by culturing in a normal 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-fusion 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 that produces the monoclonal antibody thus produced can be subcultured in a normal culture medium, and can be stored for a long 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 the secretory eIF5A protein, and a divalent antibody is also used. Valuable antibodies are also 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. , Academic Press, Inc., Pepsin, A. & Skera, A. Methods in Enzymeology (1989) 178,476-496, Academic Press, Inc., Lamoyi, E., Metan662, , 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 linking the V region, for example, any single-strand 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 a primer pair 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 produced by the host by acquiring and expressing the gene 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 (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 IF5A protein and the other 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 prepare bispecific antibody-producing fused cells. it can. Furthermore, it is also possible to prepare a bispecific antibody by a genetic engineering method.

As shown in Examples below, a neutralizing antibody against secretory eIF5A has an excellent preventive and therapeutic effect on diabetic retinopathy. The effect is superior to that of anti-VEGF drugs.
Here, with respect to diabetic retinopathy, the neutralizing antibody of the present invention specifically exhibits an action of suppressing edema of the retina and suppressing bleeding in the capillaries of the retina.
Further, when the neutralizing antibody of the present invention is used in combination with an anti-VEGF drug, the edema inhibitory effect and the bleeding inhibitory effect on diabetic retinopathy are synergistically enhanced. Therefore, the combination therapy of the neutralizing antibody of the present invention and the anti-VEGF drug is useful for the treatment of diabetic retinopathy.
Anti-VEGF agents include aflibercept, bevacizumab, ranibizumab, sodium pegaptanib and the like.

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

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 dosage forms such as ointments, inhalants, and suppositories. In injectable formulations, the neutralizing antibody can be dissolved in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks solution, Ringer 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 medicament of the present invention can be encapsulated in a polymer matrix or the like and formulated as a continuous release preparation.

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, but in general, neutralizing antibodies 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 in the range of 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)
The anti-ORAIP antibody (clone YSP5-45-36) contains amino acid residues (positions 44-72) of the human eIF5A protein (the 50th lysine residue to be hypinated and the 69th tyrosine residue to be sulfated). A peptide consisting of (including) bound to keyhole lysine 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 Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation.

Example 2
(Dissolution of CDR regions of monoclonal antibody)
Total cytoplasmic RNA was recovered from the hybridoma produced by clone YSP5-45-36.
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 consensus sequence obtained was analyzed by 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. 7, and the light chain variable region is shown in SEQ ID NO: 8 and FIG.

Example 3
(Effect on diabetic retinopathy)
In diabetic retinopathy, the disappearance of pericytes that line the walls of capillaries is thought to cause a series of vascular abnormalities. In fact, when an inhibitory antibody against platelet-derived growth factor receptor β (platelet-developed growth factor receptor β, PDGFRβ) was administered once intraperitoneally in 1-day-old mice to eliminate pericytes on the retinal vessel wall, postnatal After 8 days, edema and bleeding similar to diabetic retinopathy can be reproduced (Fig. 1, Non-Patent Document 2). By using this diabetic retinopathy model mouse, it is possible to verify the retinal edema / bleeding suppressing effect of various drugs including an anti-VEGF drug (Fig. 2). In the present invention, a single dose of anti-PDGFRβ monoclonal antibody was administered intraperitoneally to 1-day-old mice, and then the neutralizing antibody (anti-ORAIP antibody) of the present invention was intraperitoneally administered daily from 7 days after birth. It was confirmed that retinal edema and bleeding in the above were significantly suppressed (Figs. 3 and 4).

FIG. 1 shows a diabetic retinopathy model mouse used in the present invention. A single administration of an anti-PDGFRβ monoclonal antibody into the abdominal cavity of 1-day-old wild-type C57BL / 6 mice causes retinal edema and bleeding after 8 days of age due to the disappearance of pericytes on the retinal blood vessel wall. In FIG. 1, 10-day-old retinal edema / bleeding is evaluated on a 4-point scale.
Grade 1: No retinal hemorrhage / edema Grade 2: Local retinal hemorrhage or edema Grade 3: Retinal edema less than half a circumference Grade 4: Collapse of retinal tissue Figure 2 shows edema / edema of VEGF receptor decoy in diabetic retinopathy model mice. Shows inhibitory effect. A single dose of anti-PDGFRβ antibody (40 μg) was administered intraperitoneally to 1-day-old wild-type C57BL / 6 mice (5 individuals), and human immunoglobulin G (immunoglobulin G, IgG) Fc was administered to one eye on the 8th day after birth, and the opposite eye. Was administered with VEGF receptor decoy (aflibercept, 5 μg) and analyzed on the 11th day after birth.
FIGS. 3 and 4 show the edema / bleeding suppressing effect in diabetic retinopathy model mice. A single dose of anti-PDGFRβ antibody (40 μg) was administered intraperitoneally to 1-day-old C57BL / 6 wild-type mice (11 individuals), and from 7 days after birth, 5 individuals were given mouse IgG and 6 individuals were given the neutralizing antibody of the present invention (anti-ORAIP). Antibodies) (100 μg) were intraperitoneally administered daily, and both eyes of each individual were analyzed on the 11th day after birth.

Example 4
Figure 5 shows the effect of anti-VEGF drug (Aflibercept) monotherapy and anti-VEGF drug (Aflibercept) and anti-ORAIP antibody combination therapy on retinal edema / bleeding using diabetic retinopathy model mice in which pericite has disappeared. Is shown.

The experimental method is as follows.
(Experimental method using diabetic retinopathy model mice in which pericytes have disappeared)
(1) Intraperitoneal administration of 50 μg of anti-PDGFRβ antibody (clone APB5) on the 1st day after birth (2) Intraperitoneal administration of the following solution (1 μL) to each group on the 8th day after birth 1) PBS
2) Anti-VEGF drug (Aflibercept) 2 mg / mL in PBS
3) Anti-VEGF drug (Aflibercept) 2 mg / mL
＋ Anti-ORAIP antibody (clone YSP5-45-36) 9.614mg / mL in PBS
(3) Determining the grade of retinal edema / bleeding on the 13th day after birth

FIG. 5A shows a photograph of the retina of each eyeball in the three groups. FIG. 5B shows the grade of retinal hemorrhage / edema in each group. Compared to the PBS group, the anti-VEGF drug (Aflibercept) alone group has an increased proportion of retinas showing a lower grade. In the combined administration group of the anti-VEGF drug (Aflibercept) and the anti-ORAIP antibody (YSP5-45-36), the proportion of retinas showing a low grade was further increased as compared with the anti-VEGF drug (Aflibercept) alone administration group.
From the above, it was strongly suggested that the combined use of anti-ORAIP antibody with anti-VEGF drug has an additive effect on edema / bleeding suppression.

Therefore, the severity of Grades 1 to 4 was scored from 0 to 3 for convenience (Grading score), and the results of comparing the effects of monotherapy and combination therapy on PBS are shown in FIG. Grading scores for each group of PBS, monotherapy, and combination therapy are (2.86 ± 0.38 [mean ± SE], n = 7), (2.33 ± 0.33, n = 9), (1.89 ± 0.31, n = 9), respectively. The severity of the combination therapy tended to improve compared to the monotherapy.
Furthermore, when the Grading score was statistically compared, there was no significant decrease in the Grading score between the PBS-administered group and the monotherapy group (* P = 0.1864, not significant [NS]), whereas PBS-administered. A significant decrease was observed between the group and the combination therapy group (** p = 0.0296; Dunnett's multiple comparison test) (Fig. 7).
From the above, it was clarified that the combined use of the anti-ORAIP antibody with the anti-VEGF drug has an additive effect on the suppression of retinal edema and bleeding.

By using the drug or pharmaceutical composition of the present invention, the therapeutic effect on diabetic macular edema of conventionally used drugs such as anti-VEGF drugs can be enhanced. In this disease, there is a problem that recurrence occurs frequently even when the therapeutic effect is obtained by the anti-VEGF drug, and many patients are refractory to the anti-VEGF drug. Therefore, the drug of the present invention is extremely useful. high.

Claims (12)

1. A prophylactic or therapeutic drug for diabetic retinopathy containing a neutralizing antibody against secretory eIF5A as an active ingredient.
2. The prophylactic or therapeutic agent according to claim 1, wherein the neutralizing antibody against the secretory eIF5A is a monoclonal antibody.
3. The neutralizing antibody against the secretory eIF5A is heavy chain CDR1 containing the amino acid sequence shown by SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence shown by SEQ ID NO: 2, and heavy chain CDR3 containing the amino acid sequence shown by SEQ ID NO: 3. , A neutralizing antibody having a light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. The prophylactic or therapeutic agent according to claim 1 or 2.
4. Use of neutralizing antibody against secretory eIF5A for the prevention or manufacture of therapeutic agents for diabetic retinopathy.
5. The use according to claim 4, wherein the neutralizing antibody against the secretory eIF5A is a monoclonal antibody.
6. The neutralizing antibody against the secreted eIF5A 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 a weight containing the amino acid sequence shown in SEQ ID NO: 3. Neutralizing antibody with 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 use according to claim 4 or 5.
7. Neutralizing antibody against secretory eIF5A for use in the prevention or treatment of diabetic retinopathy.
8. The neutralizing antibody according to claim 7, wherein the neutralizing antibody against the secretory eIF5A is a monoclonal antibody.
9. The neutralizing antibody against the secretory eIF5A is 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 heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 3. , A neutralizing antibody having a light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. The neutralizing antibody according to claim 7 or 8.
10. A method for preventing or treating diabetic retinopathy, which comprises administering an effective amount of a neutralizing antibody against secretory eIF5A.
11. The method according to claim 10, wherein the neutralizing antibody against the secretory eIF5A is a monoclonal antibody.
12. The neutralizing antibody against the secretory eIF5A is 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 heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 3. , A neutralizing antibody having a light chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. The method according to claim 10 or 11.

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