KR20090043910A - A pharmaceutical composition comprising an anti-4-1bb antibody for preventing and treating sjogren's syndrome and lupus - Google Patents

Abstract

The present invention relates to a composition containing anti-4-1BB, an antibody effective for the prevention and treatment of Sjogren's syndrome and lupus, and specifically, to reduce the pathogenic diseases appearing in lpr mice as well as exocrine function, pathogenic CD4 ⁺ , B1 It is useful as a pharmaceutical composition for the prevention and treatment of Sjogren's syndrome and lupus because it produces regulated CD11c ⁺ CD8a ⁺ cells that significantly express IDO in secondary immune and target organs, as well as elimination of B2 and CD4 ^- CD8 ^- CD3 ⁺ B220 ⁺ Can be used.

4-1BB, Sjogren's syndrome, lupus

Description

A pharmaceutical composition comprising an anti-4-1 antibody for preventing and treating sjogren's syndrome and lupus

The present invention relates to a pharmaceutical composition for preventing and treating Sjogren's syndrome and lupus containing an anti-4-1BB antibody.

[Document 1]Foell J et al., CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB x NZW F1 mice. J Clin Invest. 2003 111 1505-18

Sjogren's syndrome is a system chronic inflammatory disease that primarily affects the lacrimal gland and salivary gland. Sjogren's syndrome is the most widely distributed autoimmune disease, accounting for 1-3% of the world's population, including 4,000,000 patients in the United States. More than 90% of patients with Sjögren's syndrome are women. The disease may be accompanied by various other autoimmune phenomena. The physiological causes of Sjögren's syndrome are not exactly understood, but two distinct phenomena are believed to occur. There are two distinct periods of initiation of the activation of autoreactive lymphoid cells and the effect of reducing the amount of tears and saliva by invading and accumulating pathogenic lymphoid cells in the lacrimal glands and salivary glands.

Although it is not yet known what causes the autoimmune response that causes lupus, previous results show that autoantibodies that respond directly to various components are the main factors.

Because of the long period between onset and effect, the early diagnosis of SCC and ongoing diagnosis of Sjogren's syndrome in humans is difficult to study. Therefore, Sjogren's syndrome and lupus model are very valuable. MRL / Mpj-Tnfrs6lpr mice develop severe autoimmune diseases similar to human Sjogren's syndrome and lupus, which require CD4 + T cells associated with the induction of autoimmune diseases. This transgenic mouse is therefore a very suitable model compared to patients with Sjogren's syndrome and lupus disease. The pathogenicity of Sjogren's syndrome and lupus in humans and rats is associated with a predominantly Th1 cytokine response. Other immunomodulators and cytokines IL-1, IL-2, IL-2R, IL-6, IFN-a and TGF-b are present in the tissues of patients with Sjogren's syndrome and lupus with severe disease.

4-1BB is a TNFR-based gene encoding a 50-55kDa protein and is expressed in CD4, CD8, NKT cells, and dendritic cells as costimulatory molecules that are induced by activation. The signal delivered to 4-1BB is preferentially received by TRAF and subsequently induces the activities of ASK-1, MAPK kinase, MAPK3 / MAPK4, p38, and JNK / stress-activated kinase, and ATF- The activity of several transcription factors such as 2, Jun, and NK-kB is induced. The importance of 4-1BB in various clinical conditions has been demonstrated through experiments using mice without the 4-1BB gene and signal interference with antibodies. The relationship between autoimmune diseases such as lupus and the 4-1BB signal has been mentioned recently (Foell J et al., CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB x NZW F1 mice. J Clin Invest. 2003 111 1505-18).

In order to clarify the importance of 4-1BB in Sjogren's syndrome with lupus disease, we made lpr mice (lpr / 4-1BB-/-) without the 4-1BB gene. As a result of observation, the lpr mice without the 4-1BB gene induced more severe autoimmune diseases and premature death. Also, the lpr mice with 4-1BB were treated with antagonistic anti-4-1BB antibodies to lpr mice, thereby not only exocrine function but also lpr mice. It has been shown to alleviate pathogenic diseases in rats, and the therapeutic effects of anti-4-1BB antibodies against Sjogren's syndrome and lupus are associated with the removal of pathogenic CD4 +, B1, B2, and CD4-CD8-CD3 + B220 + and secondary immune organs. The present invention was completed by confirming that it is associated with the selective production of regulatory CD11c + CD8a + cells that express significantly IDO in the target organ.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of Sjogren's syndrome containing an anti-4-1BB antibody as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention and treatment of lupus containing the anti-4-1BB antibody as an active ingredient.

Anti-4-1BB antibody as defined herein is characterized in that 0.1 to 50% by weight of the total weight of the composition.

Anti-4-1BB antibodies as defined herein are characterized by the removal of pathogenic CD4 +, B1, B2 and CD4-CD8-CD3 + B220 + and producing CD11c + CD8a + cells expressing IDO.

The pharmaceutical composition containing the anti-4-1BB antibody of the present invention as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

Pharmaceutical dosage forms of the composition containing an anti-4-1BB antibody of the present invention may be used in the form of their pharmaceutically acceptable salts, and may also be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection. Can be.

Compositions comprising an anti-4-1BB antibody according to the present invention are oral formulations, external preparations, suppositories, and sterile injections of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods. It can be formulated and used in the form of a solution. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the composition comprising an anti-4-1BB antibody of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition comprising the anti-4-1BB antibody of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The composition comprising the anti-4-1BB antibody of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Since the anti-4-1BB antibody of the present invention has almost no toxicity and side effects, it is a drug that can be used safely even for prolonged administration for the purpose of prevention.

Administration of the anti-4-1BB antibody of the present invention not only exocrine function, but also reduces pathogenic diseases in lpr mice, elimination of pathogenic CD4 +, B1, B2 and CD4-CD8-CD3 + B220 + and secondary immune and target organs It has been confirmed that it produces regulatory CD11c + CD8a + cells that express significantly IDO in, and thus may be useful for the prevention and treatment of Sjogren's syndrome and lupus.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  1. Preparation of Antibodies

Fusion cells producing anti-4-1BB monoantibody (3E1) were provided by Dr. Robert Mittler, Emory University, Atlanta, GA. The antibodies were also produced from ascites and fusion cell cultures and purified in the laboratory using protein G-columns (protein G-column, Sigma, St. Louis, MO). Purified mouse IgG used as an antibody in the control was purchased from Sigma-Aldrich.

Example  2. Experimental Animal

MRL / Mpj-Tnfrs6lpr mice were purchased from Jackson Laboratories. In order to make lpr mice without the 4-1BB gene, the mice were bred with the lpr mice without the 4-1BB gene. More than nine generations were backcrossed to make 90% of the MRL's background gene. Genotypes of transgenic mice were confirmed by Southern analysis and polymerase chain reaction (PCR). All rats were administered in animal-free farms without specific pathogens at the University of Louisiana's health science center. All experiments were performed using rats of matched species and age. The method of animal testing was approved by the Organized Animal Care and Use Committee of the University of Louisiana's Health Scicence Center.

Example  3. lpr Treatment of Anti-4-1BB Antibodies in Mice

Four-week-old lpr mice were injected intraperitoneally with PBS antagonist 4-1BB antibody dissolved in PBS to 200 μg per mouse. Rat IgG was injected as a control. Lpr mice without 4-1BB were injected with sterile PBS in the same manner. Mice were sacrificed 2, 3 and 4 months after starting the injection.

Example  4. Tear generation and Invasive  Measure

Tear formation was measured using cotton threads impregnated with phenol red. One end of the thread was placed on the lateral canthus of the lateral eye using tweezers and left for 5 minutes. The wet thread changed from yellow to red and its length was measured. Saliva were collected from experimental and control rats anesthetized with a diluted mixture of ketamine and xylazine. Saliva secretion was facilitated by intraperitoneal injection of 100 μl of a mixture of 0.2 mg isoproterenol and 0.05 mg pilocarpine per 100 g mouse weight. Five minutes after the injection of the mixed solution, the saliva was collected from the oral cavity using a micro pipette at an interval of 10 minutes. After centrifugation to remove drops, the amount of saliva was measured with a micropipette. In the same experiment, the total amount of protein in the saliva was measured using a BCA kit. The bovine serum albumin (BSA) protein of known quantity was used as a standard.

Example  5. Investigation of Skin Diseases and Lymph Node Tumors

Skin disease was examined monthly until the sacrifice of mice until the last five months. The extent of skin disease was determined as follows. 0, normal, 0.5, slightly (weakness in the tip of the nose and ears); 1, medium (1 or less centimeters of skin disease in the ears and nose), 2, slightly more severe (2 or less centimeters of skin disease at the tip of the nose and eye skin), and 4, severe (at the tip of the nose and eye skin) Skin disease more than 2 cm). Lymphadenopathy was assessed by swelling levels of inguinal and cervical lymphocytes (0 = normal; 1 = rudimentary; 2 = slightly larger; 3 = large, 4 = trophic).

Example  6. Immune blotting Immunoblotting )

To identify the AQP5 protein in the tears, mice were anesthetized and tears were collected using the main chamber. The tears collected from 3-5 mice were collected and transferred to a micro fuse tube containing 100 μl PBS and boiled for 5 minutes. The amount of protein from the main chamber was determined using a BCA assay. Saliva was also extracted by the method mentioned above. Lysates in the kidneys of the lacrimal glands and salivary glands were extracted with cold buffer. The lysate was centrifuged to transfer the supernatant to a new tube and stored until analysis at -80 ° C. Samples were electrophoresed on SDS-PAGE and then transferred to a PVDF membrane. Using 5% dried skin milk, the membrane was blocked and reacted with an anti-AQP5 antibody or rabbit polyclonal antipodrin antibody for 12 hours. Bound immunocomplexes were identified using an ECL reagent after treatment with an appropriate amount of HRP bound secondary antibody.

Example  7. Histological and histochemical investigation

The tissues of the sacrificed mice were planted and frozen in tissue Tek O.C.T solution. The tissue was sectioned to a thickness of 7 μm and subjected to H & E staining according to the usual method. In order to analyze the Tunnel (TUNEL) 7um thick tissue was fixed and carried out using a kit purchased from the company according to the manufacturer's method. The tissue was observed with an Eclipse E600 microscope (Olympus) and photographed. Staining of the Germinal center was performed using peanut agglutinin conjugated with biotin and an enzyme complex conjugated with avidin: biotin. All other tissue chemical staining methods were performed in the usual manner using avidin: biotin peroxidase purchased from Vector Laboratories. To remove peroxidase activity present in tissues, 0.3% hydroperoxide was treated, blocked with avidin D / biotin reagent, and nonspecific antibodies. To remove binding, 5% rabbit serum was treated with TBS buffer. The primary antibody was reacted at 4 ° C. for 12 hours and treated with antirat-IgG (H + L) conjugated with biotin diluted to 100% for 1 hour. Diaminobenzidine reagent (reagent) was used to show the color of immunostaining, and tissues were stored after H & E staining was performed together. The tissue was observed with an Eclipse E600 microscope and photographed.

Example  8. Immunofluorescence Staining

The 7 μm sectioned tissue was dried and fixed in cold acetone and dehydrated in TBS before being used for the experiment. After blocking with serum of 5% normal rabbit, FITC-bound goat anti-mouse IgG and FITC-coupled goat anti- IgG fragment of complement receptor 3 were reacted for 1 hour at room temperature. It was. Tissues were stored after washing three times with TBS containing 0.05% Tween20. The tissue was observed with an Eclipse E600 microscope and photographed.

Example  9. Antigen Specific Recall Reactions

Cells isolated from draining lymph nodes (2 × 10 ⁵ / well) were round-bottomed for 96 hours in a medium containing recombinant anti-fodrin at a concentration of 5 μg / ml. Incubated in microtiter plates. As a control, cells were treated with 5 ug / ml ConA, 5 μg / ml OVA, and 5 μg / ml KLH. Alamar blue was treated 16 hours before measuring the proliferation of the cells, and the degree of proliferation of the cells was measured according to the manufacturer's method.

Example  10. Flow cell  analysis

To identify the phenotype of lymphoid cells, 1 μg of anti-FcgR antibody was first treated and analyzed using flow cytometry (Becton-Dickinson FACS caliber, flow cytometry) and CellQuest software. Anti-CD3e, CD4, CD5, CD8a, CD11c, and B220 antibodies were used. Draining lymphocytes were used without additional separation or by reaction with anti-CD11c microbeads and then separated using a MACS column. The surface of the cells was stained and permeable using FITC-bound antibodies, then reacted with PE-bound IFN-γ, and cell separation and flow interferon gamma (IFN-γ) expression by flow cytometry. This was analyzed.

Example  11. Determination of the amount of serum antibody and uptake in vivo

Serum samples were obtained from 3 and 5 month old lpr mice treated with IgG and anti 4-1BB antibodies and lpr mice without the 4-1BB gene, respectively. Annually diluted serum was analyzed for the amount of Ig by ELISA. Total amounts of IgG1, IgG2a, IgG2b were analyzed using the kit according to the manufacturer's method. Analysis of anti DNA specific antibodies was performed by adding serially diluted serum to a microtiter treated with 10 μg / ml of dsDNA. HRP activity was developed using TMB substrate and absorbance was measured at 450 nm. Antinucleolar antibodies present in serum were measured using a slide treated with HEp-2. The functional ability of the marginal zone was confirmed using polysaccharides bound to the fluorescent material. To confirm this, 200 μl of 500 kDa lysine immobilized dextran-FITC was injected into the vein in the tail of the rat. After 40 minutes, the rats were sacrificed and the spleen was taken out and placed in a tissue-T solution. The tissue thus prepared was used for histochemical analysis.

Example  12.RT- PCR Analysis of IDO by

Total RNA was isolated from lacrimal glands and salivary glands using TRIZOL. The expression of specific IDO mRNA was measured by RT-PCR. Oligo (dT) with 2μg of RNA (oligo (dT)) using a _10-15 primer (primer) to make cDNA and using the primers shown in Table 1 below to confirm the expression of IDO. The resulting PCR result was confirmed by staining with EtBr after electrophoresis on 1% agarose gel (agarose gel).

 protein Primer Sequence   IDO Sense: SEQ ID NO: 1 5'-CACTGTACCAGTGCAGTAG-3 ' Anti-sense: SEQ ID NO: 2 5'-ACCATTCACACACTCGTTAT-3 '

Example  13. Reebok Nuclease  Protection analysis ( RNase  protection assay, RPA )

Total RNA was isolated from lacrimal glands and salivary glands using TRIZOL. Expression of cytokine and chemokine mRNAs was confirmed by RPA performed according to the manufacturer's method (BD RiboQuant ™ Ribonuclease Protection Assay Systems). 10 μg of RNA was reacted at 65 ° C. with a probe containing a-32P UTP to which radioactivity was attached. After the reaction, unbound ssRNA was removed by RNase treatment and dsRNA was purified by phenol / chloroform extraction and precipitated by ethanol treatment. The prepared samples were electrophoresed on 6% polyacrylamide gel, transferred to 3mm paper, and dried to be exposed to the film.

Experimental Example  1. Effect of 4-1BB Gene Deficiency on Infiltration of Leukocytes in the Lacrimal and Salivary Glands

1-1. Experiment method

To investigate the importance of 4-1BB gene in autoimmune diseases including lacrimal glands and salivary glands, experiments were conducted on lpr mice without 4-1BB gene. In addition, antagonist 4-1BB antibodies were treated with lpr mice that normally had 4-1BB gene. Lpr / 4-1BB + / + mice treated with IgG and an anti-CD137 antibody and lpr / 4-1BB − / − mice treated with PBS were sacrificed at 5 months (4 post-treatment). Months). After measuring the size of lacrimal glands and salivary glands (FIG. 1A) and the weights of lacrimal glands and salivary glands (FIG. 1B), the lacrimal glands and salivary glands were prepared by treating collagenase / Dnase I (collagenase / Dnase I). 1C). The prepared single cell suspension was blocked by treatment with anti-FcgR antibody and then subjected to two-color flow cytometry. The numbers shown in each zone represent the percentage of the displayed cell population (FIGS. 1D and 1E below). The lacrimal glands and salivary glands were separated, frozen in tissue-Tek solution, the tissues were sliced to a thickness of 7 μm, and subjected to H & E staining for histological analysis on CD4 and CD8a.

1-2. Experiment result

As a result of the experiment, the lpr mice without the 4-1BB gene did not increase significantly in the size of the tissues compared to the lpr mice treated with the rat IgG, but the size of the tissues was significantly reduced in the lpr mice treated with the anti-4-1BB antibody. Was observed. Lpr mice treated with anti- 4-1BB antibody showed significantly reduced CD4 and CD8a cell infiltration into the lacrimal glands and salivary glands compared to lpr mice treated with IgG. Lpr mice without the 4-1BB gene show a slight increase in leukocyte infiltration into the lacrimal glands. For salivary glands, a significant increase in CD4 + cells can be seen in comparison with IgG treated controls and some evidence for CD8 + cells (Figure 1C below). Mice treated with anti-4-1BB antibodies showed markedly reduced infiltration of leukocytes into the lacrimal glands and salivary glands (FIG. 1 C, D, E). Therefore, it was confirmed that 4-1BB plays an important role in the development of autoimmune diseases by affecting the movement of leukocytes that cause Sjogren's syndrome.

Experimental Example  2. Effect of Lack of 4-1BB Gene on the Function of Tears

2-1. Experiment method

AQP5 secretion is used as a diagnostic marker for loss of function in the lacrimal glands of lpr mice. Tears were collected from lpr / 4-1BB + / + treated with 5-month old IgG, lpr / 4-1BB + / + treated with anti-4-1BB antibody, and lpr / 4-1BB − / − mice to collect expression of AQP5 protein. Investigate. The amount of tears by measuring the wet length of cotton thread in lpr / 4-1BB + / + mice treated with IgG and anti-4-1BB antibodies aged 1 to 5 months and lpr / 4-1BB-/-mice treated with PBS. Was measured (FIG. 1A). Proteins and lacrimal lysates (FIG. 1C) obtained from tears of 5 months old rats were electrophoresed at 20 μg each in reducing conditions, and then transferred to PVDF membranes. The expression was confirmed using probes against AQP5 and anti-a-fodrin antibodies. .

2-2. Experiment result

As a result of the experiment, the expression of AQP5 protein in lpr / 4-1BB-/-was higher than that in IgG-treated lpr / 4-1BB + / + mice, whereas lpr / 4 treated with anti-4-1BB antibody was observed. Expression of AQP5 protein was not observed in −1BB + / + mice (FIG. 2B). Similarly, anti-fordrin (a-fordrin) of 120 kDa fragments were identified in the lacrimal glands of lpr / 4-1BB-/-mice, indicating the degree of loss of lacrimal glands (FIG. 2C). In lpr / 4-1BB + / + mice treated with anti-4-1BB antibody, AQP5 was not expressed in tears, and in the lacrimal gland, no a-fordrin of 120 kDa fragment was identified (FIGS. 2B and 2C). Therefore, lpr mice lacking 4-1BB showed a slightly decreased lacrimal gland function.

Experimental Example  3. Effect of 4-1BB Gene Deficiency on Salivary Gland Function

3-1. Experiment method

Saliva were collected using micropipettes in lpr / 4-1BB + / + mice treated with IgG and anti4-1BB antibodies aged 1 to 5 months and lpr / 4-1BB − / − mice treated with PBS. The bubbles were removed by centrifugation and the amount of saliva produced was examined. Salivary glands were isolated from 5 month old mice, and lysates were prepared and electrophoresed on 12% SDS-PAGE under reducing conditions. 20 ug of protein per electrophoresis was transferred to the PVDF membrane by electrophoresis, and polyclonal a-fodrin antibody was probed for expression. Arrows indicate the relative movement of fragmented anti-fodrin subunits (FIG. 1D), fixing frozen sections (7um) of salivary glands and identifying TUNEL + apoptotic cells using the kit. . Draining lymph node cells (2 × 10 ⁵ / well) were incubated for 96 hours in a medium containing recombinant antipodrin (a-fodrin, 5ug / ml) or other labeled reagents (all 5ug / ml).

3-1. Experiment result

As a result of the above experiments, it was confirmed that lpr / 4-1BB-/-mice produced significantly reduced saliva, even surprisingly even under induction conditions (FIG. 3A). This is a very important observation, as previously reported results show that lpr mice can produce reduced saliva only in non-induced conditions. In addition, we examined the loss of salivary gland function in lpr / 4-1BB-/-mice by investigating the truncated form of a-fodrin, a protein for autoantibodies produced in patients with Sjogren's syndrome and experimental animals. As a result, an increased 120-kDa fragment a-fordrin was confirmed in the lysate prepared in the salivary glands of lpr / 4-1BB-/-mice (FIG. 3B). The expression of a-fodrin of 120 kDa fragments in lpr / 4-1BB + / + and lpr / 4-1BB − / − treated with IgG showed similar expression (FIG. 3C). The identification of 120-kDa fragment a-fordrin from 240-kDa a-fodrin is known as a result of apoptosis. Consistent with these results, more TUNEL-positive apoptotic cells in salivary glands were identified in lpr / 4-1BB-/-mice compared to lpr / 4-1BB + / + (FIG. 3D). These findings demonstrate the severe damage of salivary glands seen in lpr / 4-1BB-/-mice, indicating an increased apoptotic mechanism dependent on Fas in lpr / 4-1BB-/-mice. To investigate the differences seen in lpr / 4-1BB + / + treated with IgG, lpr / 4-1BB + / + treated with anti-4-1BB antibody and lpr / 4-1BB − / − mice, recombinant a-fordrin was examined. Recall response to lpr / 4-1BB + / + treated with anti 4-1BB antibody showed weak cell proliferation against a-fordrin (FIG. 3E), whereas lpr / 4 Significantly proliferative responses were seen in −1BB − / − mice. IgG-treated lpr / 4-1BB + / + mice also showed proliferative responses similar to lpr / 4-1BB − / −. Thus, severe salivary gland dysfunction was identified in lpr mice lacking 4-1BB.

Experimental Example  4. Effect of 4-1BB Gene Deficiency on Skin Disease, Lymph Node Tumor and Mortality

4-1. Experiment method

Representative phenomena of autoimmune diseases in lpr mice are skin diseases in the back, neck and ears as well as enlarged spleen and lymph nodes. We investigated the pathological symptoms of damage to the exocrine glands in lpr / 4-1BB-/-and the suppression of disease seen in lpr / 4-1BB + / + mice treated with anti-4-1BB antibodies. The skin diseases seen in lpr / 4-1BB + / + mice treated with 5-month old IgG and anti-4-1BB antibody and lpr / 4-1BB-/-mice treated with PBS were examined. Changes in body weight were determined up to 5 months old, and spleen and lymph nodes were isolated from each other. The weights of spleen and lymph nodes isolated from 5 months old rats were measured (figure G). Mortality was assessed monthly in lpr / 4-1BB + / + mice treated with 1BB antibody and lpr / 4-1BB − / − mice treated with PBS.

4-2. Skin disease result by 4-1BB gene deficiency

No skin disease was seen in lpr / 4-1BB + / + mice treated with anti-4-1BB antibody (FIGS. 4A and B). In IgG-treated lpr / 4-1BB + / + mice, skin diseases were observed around the nose and ears. In lpr / 4-1BB-/-mice, 60-80% of 4-month-old mice had severe ear tips. Not only did they disappear, but serious skin diseases were shown (FIGS. 4A and 4B).

4-3. Lymph node tumor result by deficiency of 4-1BB gene

The lpr / 4-1BB-/-mice began to become significantly heavier than the lpr / 4-1BB + / + treated with IgG at 3 months of age, while the lpr / 4-1BB-/-mice had lpr / 4 treated with IgG. Much lighter than -1BB + / + (FIG. 4C below). To account for the increase in body weight seen in lpr / 4-1BB-/-mice, we examined the possibility that secondary lymphoid tissue was enlarged and compared with lpr / 4-1BB + / + treated with lpr / 4-1BB. -/-In the rats, apparently enlarged lymph nodes were identified at 3 months and significantly increased lymphadenopathy was observed at 4 and 5 months of age. No symptoms of lymphadenopathy were seen in lpr / 4-1BB + / + mice treated with anti-4-1BB antibody (FIG. 4D). In lpr / 4-1BB-/-mice, significantly larger lymph nodes (specifically inguinal and mesenteric lymph nodes) were identified compared to IgG-treated lpr / 4-1BB + / +. In lpr / 4-1BB + / + mice treated with 1BB antibody, lymphadenopathy induced by lpr mutation was completely blocked (FIGS. 4E and 4F).

4-4. Mortality due to deficiency of the 4-1BB gene

Survival rates of lpr / 4-1BB + / + and lpr / 4-1BB − / − mice were examined to determine whether deficiency of the 4-1BB gene affected mortality in lrp mice. The lpr / 4-1BB-/-mice were almost dying at 4 months and showed significantly reduced motility due to lymphadenopathy and only 20% survived at 5 months. All lpr / 4-1BB + / + mice treated with IgG died at 8 months of age. All lpr / 4-1BB + / + mice treated with anti-4-1BB antibody were all alive up to 10 months. Overall, these results confirmed that 4-1BB is important for the survival of lpr mice with autoimmune diseases.

Experimental Example  5. Effects of 4-1BB Gene Deficiency on the Production of Autoantibodies

5-1. Experiment method

One important aspect of the relationship between disease and lpr mutations is the production of autoantibodies and the accumulation of Ig in target organs. Serum was collected by the puncture of the heart and autologous nucleic acid (necleolar) antibody production was confirmed using a slide attached with HEp-2.

Rats were sacrificed at 3 and 5 months of treatment with IgG and anti 4-1BB antibody, diluted as indicated in serum and prepared for analysis of titer of anti-nucleic acid antibody in serum. Was added to the attached slide. Bound immune complexes were shown using anti IgG antibody with FITC attached. Serum obtained from 5 month old rats was added to a microplate attached to anti-IgG and the concentration of autoantibodies was examined using an ELISA kit. Serum obtained from 5 month old mice was added to dsDNA treated microplates and immune complexes were confirmed by ELISA method.

5-2. Experiment result

Autologous nucleic acid production was not observed in lpr / 4-1BB + / + mice treated with anti-4-1BB antibodies at 3 months and 5 months, but lpr / 4-1BB + / treated with IgG in lpr / 4-1BB − / − mice. Significantly increased autologous nucleic acid antibody production compared to mice (FIG. 5A). In the present application, antibody titers of serum were confirmed at various dilution ranges. In the most diluted concentrations (1: 2500), IgG-treated lpr / 4-1BB + / + mice did not identify antibodies, but lpr / 4 In the case of -1BB-/-mice, although weak, the production of antibodies was confirmed (FIG. 5B). Analysis of the total IgG and anti-DNA antibody production in serum showed that the amount of IgG and the production of anti-DNA antibody were more evidenced when compared to lpr / 4-1BB + / + in lpr / 4-1BB − / − mice. In the lpr / 4-1BB + / + mice treated with anti-4-1BB antibodies, the amount of serum antibodies was significantly reduced (FIGS. 5C and 5D).

Experimental Example  6. Effect of 4-1BB Gene Deficiency on B Cell Function

6-1. Experiment method

Based on the amount of increased serum antibodies identified in lpr / 4-1BB-/-, the number and function of B cells were examined in lpr / 4-1BB-/-.

Frozen sections of spleens of lpr / 4-1BB + / + mice treated with 5-month old IgG and anti-4-1BB antibodies and lpr / 4-1BB − / − mice treated with PBS were reacted with biotin-bound PNA. Then streptavidin-HRP was added. Diaminobenzidine substrate was added to make the color appear and hematoxylin was stained. Dark reaction product means PNA activity (FIGS. 6B and 6D). The spleens were treated with collagenase / Dnase I to prepare single cell suspensions. Cells in the abdominal cavity were collected using ice-cold HBSS. Samples from which red blood cells were removed were subjected to flow cytometry using specific antibodies to identify individual cell populations. Numbers in each section represent the percentage of cells in each indicated cell population (FIG. 6C) and mice were injected intravenously with dextran with FITC bound. After 40 minutes, the spleen was removed, placed in a tissue-Tek solution and frozen, and sections were made and checked under a fluorescence microscope.

6-2. Experiment result

The number and size of germinal centers identified by PNA reactivity were increased compared to lpr / 4-1BB + / + mice treated with IgG in lpr / 4-1BB − / −, as expected. In the lpr / 4-1BB + / + mice treated with the 4-1BB antibody, the formation of the low terminal center was not observed (FIG. 6A). Part of B cells in the spleen and peritoneal cavities to determine whether the number of B cells increases in lpr / 4-1BB − / − mice and decreases in lpr / 4-1BB + / + mice treated with anti-4-1BB antibodies. The subsets showed that the percentages of B1 cells (B220 + CD5 +) and B2 cells (B220 + CD5-) in lpr / 4-1BB − / − mice were treated with lpr / It was slightly higher than the spleen of 4-1BB + / + mice, but markedly different in the abdominal cavity. Lpr / 4-1BB + / + mice treated with anti-4-1BB antibody showed significantly lower percentages of B1 and B2 cells in both the spleen and the abdominal cavity (FIG. 6B).

Functional analysis was also performed to determine the antigen uptake capacity of the marginal sinus of the rat's spleen. Under normal conditions, dextran-FITC is not a marginal zone metallophilic marcrophage but an outer zone that is highly specific for neutral polysaccharides such as dextran and ficoll. It is easily uptaken by marginal zone marcrophage. As a result, the degree of dextran-FITC uptake in lpr / 4-1BB-/-and IgG-treated lpr / 4-1BB + / + mice was significantly similar and increased, and the anti-4-1BB antibody was increased. Was significantly reduced in the lpr / 4-1BB + / + treated mice (FIG. 6C). In addition, splenic, lacrimal and salivary glands compared to lpr / 4-1BB + / + mice treated with anti-4-1BB antibodies in lpr / 4-1BB-/-mice but not lpr / 4-1BB + / + mice treated with IgG. In B220 + CD3 + cells were confirmed to increase (Fig. 6D). Thus, the overreactivity of B cells in lpr / 4-1BB − / − mice and the reduced number and function of B cells in lpr / 4-1BB + / + mice treated with anti-4-1BB antibodies may be attributed to humoral responses. By showing the difference in, it was confirmed that the B cell function is increased in mice lacking 4-1BB.

Experimental Example  7. Cytokines by the 4-1BB Gene and Chemokines  Expression Pattern Analysis

7-1. Experiment method

It has long been known that disease is aggravated by increased CD4 + cell infiltration and Th1 type cytokines in lpr mice. To find possible causes of different pathogenic symptoms in lpr / 4-1BB-/-and lpr / 4-1BB + / +, cytokine and chemokine expression patterns were analyzed in lacrimal and salivary glands of 5-month-old rats, respectively. . The lacrimal glands and salivary glands were frozen with liquid nitrogen, RNA was extracted using Trizol, and RPA analysis was performed immediately.

7-2. Experiment result

As a result, IgG-treated lpr / 4-1BB + / + mice showed more cytokine and chemokine expressions in lpr / 4-1BB − / − mice, but they were expressed at similar levels overall. Increased expression of TGF-b3 in salivary glands of lpr / 4-1BB + / rats. The expression of LT-b and IFN-γ was higher in lpr / 4-1BB-/-mice than in IgG-treated lpr / 4-1BB + / + mice, and the expression of Th10 type IL10 was lpr / 4- It was confirmed that the salivary glands of 1BB-/-mice were significantly increased. Cytokines and chemokines were commonly decreased in lpr / 4-1BB + / + mice treated with anti-4-1BB antibody, which was confirmed by decreased leukocyte infiltration and preservation of salivary glands and lacrimal glands. 7). Although both Th1 and Th2 type cytokines were expressed in salivary glands and lacrimal glands of lrp mice with or without 4-1BB gene, little expression was induced in lpr / 4-1BB + / + mice treated with anti-4-1BB antibody. .

Experimental Example  8. 4-1BB Gene CD11c + CD8a + Cell and IDO Expression Analysis

8-1. Experiment method

The CD4 + T-cells that make IFN-γ are known to induce serious diseases in the Sjogren's syndrome model, lpr mice and lupus models. In this study, IFN-γ was induced in the draining lymph nodes of three experimental mice. The ability of CD4 + T cells to make was confirmed.

Single cell suspensions prepared in lymph nodes were reacted with brefeldin for 6 hours. Intracellular IFN-γ was confirmed using a flow cytometer. The numbers in each section represent the percentage of CD4 + and CD8a cells producing IFN-γ (Figure D). The surface of the cells was labeled using specific antibodies. CD11c + cells were isolated using anti-CD11c microbeads. The amount of intracellular IFN-γ was confirmed. Expression of IDO in lacrimal glands and salivary glands of lpr / 4-1BB + / + mice treated with IgG and anti-4-1BB antibodies was confirmed by RT-PCR analysis.

8-2. CD11c + CD8a IFN-γ expression in + cells

In the experiments, IFN-γ-producing cells were confined to CD4 + T cells in lpr / 4-1BB + / + mice treated with IgG, whereas CD4 + T cells and CD8a + cells in lpr / 4-1BB − /-mice. Produced similar levels of IFN-γ, and the percentage of CD4 + T cells was significantly reduced in lpr / 4-1BB + / + mice treated with anti 4-1BB antibody and significantly lower than other groups. Only cells were found to make IFN-γ. Significant amounts of IFN- [gamma] were produced by CD8a + cells in lpr / 4-1BB + / + mice treated with anti 4-1BB antibody, and the expression level was significantly higher than in other groups (FIG. 8A).

CD11c + CD8a + in draining lymph nodes, lacrimal glands and salivary glands of three different experimental rats to demonstrate whether this model is related to CD11c + CD8a + cell induction and therapeutic effects of anti-4-1BB antibodies in Sjogren's syndrome. It was confirmed that cells were generated. Although not confirmed in other groups in this experiment, CD11c + CD8a + cells were identified in lpr / 4-1BB + / + treated with anti 4-1BB antibody (FIG. 8B). As a result of testing whether these cells produce IFN-γ, it was confirmed that IFN-γ is locally expressed in CD11c + CD8a + cells (FIG. 8D).

8-3. lpr Expression of IDO in Tears and Salivary Glands of 4-1BB + / + Mice

As a result of the experiment, it was confirmed that the treatment of the anti-4-1BB antibody selectively induced the expression of IDO in the lacrimal glands and salivary glands (FIG. 8E). Expression of CD11c + CD8a + cells and IDO in lpr / 4-1BB + / + mice and lpr / 4-1BB − /-mice treated with IgG was not confirmed. The therapeutic effect seen in / + is shown to be caused by CD11c + CD8a + cells. The anti- 4-1BB antibody treatment effected in a model of mice with Sjogren's syndrome appears to be due to CD11c + CD8a + expressing high concentrations of IFN-γ and IDO.

Experimental Example  9. Effects of 4-1BB Gene Deficiency on Kidney Disease

9-1. Experiment method

Lpr mice have been studied as a model for lupus because they usually cause death from kidney disease. Therefore, we investigated whether deficiency of the 4-1BB gene in lpr mice is associated with kidney disease. Kidney size and weight in 5-month-old IgG-treated lpr / 4-1BB + / + mice, lpr / 4-1BB-/-mice, and antipractic 4-1BB antibodies-treated lpr / 4-1BB + / + mice It was. The amount of protein secreted into urine samples of rats aged 3 to 5 months was determined using commercially available reagent strp as described in the method. Frozen sections of the kidney were subjected to H & E staining and histochemical analysis for CD4 and CD8a. A-fodrin was identified in the lysate of the kidney, and the arrow indicates the relative movement of fragmented anti-fodrin proteins (FIG. 9G), and the frozen sections were stained using the FITC conjugated antibody shown in the figure. It was.

9-2. Experiment result

The kidneys of IgG-treated lpr / 4-1BB + / + and lpr / 4-1BB-/-mice were significantly larger and heavier, whereas in lpr / 4-1BB + / + treated with anti-4-1BB antibodies, It was found to be quite small (FIGS. 9A and 9B below). Kidney function in lpr mice is examined by determining the amount of protein secreted in urine. From one month old rats, we examined the protein secreted in urine every month. Significantly higher concentrations of protein were found in urine in lpr / 4-1BB-/-mice compared to lpr / 4-1BB + / + mice and in urine in 4-1BB antibody-treated lpr / 4-1BB + / + mice. The amount of secreted protein was increased to a minimum (Figure 9C below). Histological analysis showed increased glomerular infiltration (FIG. 9D) and accumulation of CD4 + and CD8a + cells, with minimal infiltration in lpr / 4-1BB + / + treated with 4-1BB antibody ( Figure 9E). As in the lacrimal glands and salivary glands, 120-kDa fragments of a-fordrin were observed in kidney lysates of 5-month old rats. In the lpr / 4-1BB + / + mice treated with IgG, a weakly expressed 120-kDa fragment of a-fordrin was observed. It was not confirmed in lpr / 4-1BB + / + (FIG. 9F). In addition, by examining the accumulation of IgG and complement receptor 3, pathological diseases of the kidneys were examined. As a result, significant IgG and complement receptor 3 were detected in IgG-treated lpr / 4-1BB + / + mice and lpr / 4-1BB-/-mice. By confirming the accumulation (FIG. 9G), the deficiency of 4-1BB gene in lpr mice was found to be related to kidney disease as well as exocrine glands. Lpr / 4-1BB + / + treated with the anti 4-1BB antibody showed significantly lower levels of IgG and complement receptor 3 accumulation.

Hereinafter, an example of the preparation of a pharmaceutical composition comprising the composition of the present invention will be described, but the present invention is not intended to limit the present invention but is only specifically intended to be described.

Formulation example  1. Preparation of Injection

Anti-4-1BB Antibody ......................................... 100 mg

Sodium metabisulfite .................. 3.0 mg

Methylparaben ...... 0.8 mg

Propylparaben ......................................... 0.1 mg

Sterile Distilled Water for Injection ...

The above ingredients are mixed and prepared in a conventional manner to have a final volume of 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation example  2. Pilled  Produce

Anti-4-1BB Antibody ......................................... 120 mg

Corn starch ... 100 mg

Sterilized Distilled Water ...............

The above components are mixed and refilled in a 0.3 cm diameter by a conventional pill manufacturing method to prepare pills.

Formulation example  3. Preparation of Tablets

Anti-4-1BB Antibody ............................................... 200 mg

Lactose ... 100 mg

Starch ..................... 100 mg

Magnesium Stearate ...............

A tablet is prepared by mixing and tableting the above components according to a conventional tablet manufacturing method.

Formulation example  4. Preparation of Capsules

Anti-4-1BB Antibody ......................................... 100 mg

Lactose ... .50 mg

Starch ..................... .50 mg

Talc .................. ..2 mg

Magnesium stearate .....................................

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  5. Liquid  Produce

Anti-4-1BB Antibody ......................................... 1000 mg

Sugar................................................. ... 20 g

Isomerized sugar ... 20 g

Lemon scent ...... .............

Purified water was added to make a total of 1000 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

1 is a diagram showing the degree of recovery of lacrimal glands and salivary glands of mice treated with IgG and anti 4-1BB antibody (anti-CD137 antibody),

2 is a diagram illustrating the function of the lacrimal glands in mice lacking 4-1BB (anti-CD137 antibody).

3 is a diagram confirming the loss of salivary glands in mice lacking 4-1BB (anti-CD137 antibody),

4 is a diagram illustrating skin disease, lymphadenopathy and premature death in mice lacking 4-1BB (anti-CD137 antibody).

5 is a diagram observing the increased production of autoantibodies in mice lacking 4-1BB (anti-CD137 antibody),

6 is a diagram confirming the increase of B cell function in mice lacking 4-1BB (anti-CD137 antibody),

Figure 7 Expression of cytokines and chemokines in exocrine glands of Ipr / 4-1Bb + / + mice treated with IgG and anti-4-1BB antibodies (anti-CD137 antibody) and Ipr / 4-1BB − / − mice treated with PBS Is also confirmed

8 is a diagram confirming the correlation between the therapeutic effect of the anti- 4-1BB antibody (anti-CD137 antibody) and the expression of IDO CD11c + CD8a + cells,

9 is a diagram confirming that the deficiency of the 4-1BB gene in Ipr mice induces kidney disease.

<110> KWON, Byoung Se <120> A pharmaceutical composition comprising an anti-4-1BB antibody          for preventing and treating sjogren's syndrome and lupus <130> DIF / 2007-05-0007 / JY <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> sense <400> 1 cactgtacca gtgcagtag 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> anti-sense <400> 2 accattcaca cactcgttat 20  

Claims (4)

A pharmaceutical composition for preventing and treating Sjogren's syndrome containing an anti-4-1BB antibody as an active ingredient. A pharmaceutical composition for the prevention and treatment of lupus containing anti-4-1BB antibody as an active ingredient. According to claim 1, wherein the anti-4-1BB antibody is a pharmaceutical composition, characterized in that 0.1 to 50% by weight of the total weight of the composition. The pharmaceutical composition of claim 1, wherein the anti-4-1BB antibody produces CD11c + CD8a + cells expressing IDO and elimination of pathogenic CD4 +, B1, B2 and CD4-CD8-CD3 + B220 +.

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