JP2009203161A - Rheumatism-treating agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a rheumatism-treating agent based on a totally new action mechanism. <P>SOLUTION: This rheumatism-treating agent comprises (A) a dendritic cell stimulated by an antigen originated from a vascular endothelial cell or (B) an antigen originated from a vascular endothelial cell and a pharmaceutically acceptable adjuvant. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to rheumatoid immunotherapy targeting neovascular endothelial cells of rheumatoid arthritis.

Rheumatoid arthritis is an autoimmune disease of unknown cause, and the progression of this disease is first the proliferation of synovial cells covering the inner surface of the joint cavity and then the blood vessels of the joints. Later, leukocytes migrate from the blood vessels to the synovial tissue of the joint, an immune response occurs locally in the joint, an inflammatory reaction is caused by the action of cytokines produced by leukocytes, and cartilage / bone destruction progresses, finally Joints are destroyed, leading to advanced dysfunction. Pain due to joint dysfunction and deformation extremely reduces the patient's QOL (Non-patent Document 1). For the treatment of rheumatoid arthritis, non-steroidal anti-inflammatory drugs, disease-modifying anti-rheumatic drugs, methotrexate, gold preparations, etc. are used (Non-patent Documents 2 and 3), but the fundamental treatment has not been established yet. The development of therapeutic drugs is urgently needed.
Koch AE, Angiogenesis: implications for rheumatoid arthritis, Arthritis Rheum, 41, 951-962, 1998 Today's Therapeutics 2006, Hiromi Mizushima, Nanedo, p285-290, 2006 edition Arthritis Research & Therapy, vol.6, No.2, p60-72 (2004)

  An object of the present invention is to provide a therapeutic agent for rheumatism based on a completely new mechanism of action.

Angiogenesis is mainly observed at the wound healing site in adults, but when focusing on the disease, it is observed at the synovial site during rheumatoid arthritis. That is, as the synovial tissue increases, blood vessels are actively regenerated in order to supply oxygen and nutrients to the synovial cells (Non-patent Document 3).
Therefore, the present inventor has paid attention to the formation of blood vessels accompanying the increase in synovial tissue, and as a result of various studies, the present inventors have investigated dendritic cells stimulated with antigens derived from vascular endothelial cells, or antigens derived from vascular endothelial cells and adjuvants. When the combination is used, humoral immunity or cellular immunity targeting neovascular endothelial cells accompanying the increase in synovial tissue is induced, and the neovascularization of the synovial tissue can be broken down by the immune mechanism. As a result of suppressing the proliferation of the tissue, it was found that a rheumatic lesion was cured, and the present invention was completed.

That is, the present invention provides a therapeutic agent for rheumatism comprising (A) dendritic cells stimulated with an antigen derived from vascular endothelial cells, or (B) an antigen derived from vascular endothelial cells and a pharmaceutically acceptable adjuvant. Is.
The present invention also provides (A) dendritic cells stimulated with an antigen derived from vascular endothelial cells, or (B) an antigen derived from vascular endothelial cells and a pharmaceutically acceptable adjuvant for producing a therapeutic agent for rheumatism. It is intended to provide use.
Furthermore, the present invention relates to a rheumatism characterized by administering (A) a dendritic cell stimulated with an antigen derived from vascular endothelial cells, or (B) an antigen derived from vascular endothelial cells and a pharmaceutically acceptable adjuvant. It provides a cure.

  The features of the therapy of the present invention can be broadly classified into the following two. Due to this feature, the following remarkable effects can be obtained and the side effects are low, and it becomes a revolutionary therapeutic agent for rheumatism.

(1) Targeting neovascular endothelial cells in the joint synovial region:
Since the site where angiogenesis is actively performed in the living body is mainly limited to the wound healing site, when targeting neovascular endothelial cells, the selectivity to the lesion tissue is extremely high, and the normal tissue Has little effect on the side effects and low side effects. Another is that the cells constituting the rheumatic lesion tissue are vascular endothelial cells: tissue cells = 1: several tens to several hundreds, which is one vascular endothelial cell from several tens to several hundreds of tissue cells. The death of one vascular endothelial cell induces the death of tens to hundreds of tissue cells. Therefore, rather than targeting the rheumatoid lesion tissue itself, the vascular endothelial cell of the tissue is targeted and destroyed, and the regression effect of the rheumatic lesion tissue can be obtained extremely effectively.

(2) Utilizing immune mechanism:
It is mentioned that the selectivity of the effector to the target, such as antibodies by humoral immunity and cytotoxic T cells in cellular immunity, is extremely high, and as a result, remarkable effects are obtained and side effects are low. Furthermore, the immune mechanism has immune memory, and it works for a very long time as compared with drug therapy, and does not require frequent administration, and can prevent not only therapeutic effects but also recurrence after treatment.

  The therapeutic agent for rheumatism of the present invention elicits immunity, and the elicited immunity targets neovascular endothelial cells in the human synovial region. Therefore, the vascular endothelial cell serving as an antigen is not particularly limited as long as it is derived from human. An antigen-pulsed dendritic cell may be immunized, or the antigen may be mixed with an adjuvant for immunization.

  Two types of vascular endothelial cells can be used, one of which is a vascular endothelial cell isolated from a human, preferably a patient. For example, vascular endothelial cells such as umbilical vein, umbilical artery, aorta, pulmonary artery, neonatal foreskin, adult skin, and adipose tissue are used. In particular, as adipose tissue is removed during cosmetic surgery, even if it is removed from a normal living body, there is no significant effect. In addition, the specific gravity of vascular endothelial cells and adipose cells in the adipose tissue is greatly different. By simply operating, vascular endothelial cells can be isolated and cultured with high purity relatively easily. Vascular endothelial cells of the patient's skin can also be used, but the amount is small and needs to be significantly expanded by culture.

The other is general cultured human vascular endothelial cells. It is not necessary to inoculate the patient's body with vascular endothelial cells themselves as antigens, but extracts from vascular endothelial cells are used as antigens, so there is a need for MHC compatibility (Major Histcompatibility Complex) Absent. Therefore, commercially available human cultured vascular endothelial cells can be used. Examples of cultured human vascular endothelial cells include umbilical vein vascular endothelial cells (HUVEC), neovascular foreskin / adult skin-derived microvascular endothelial cells (HMVEC), pulmonary artery vascular endothelial cells (HPAEC), aortic vascular endothelial cells (HAEC) and the like. It is done. Since it is derived from humans, the risk of virus transmission is not zero. Therefore, it is necessary to take appropriate measures for virus removal (heat treatment, organic solvent and surfactant treatment, special fine filtration, etc.). The medium for vascular endothelial cell culture is not particularly limited. For example, a medium such as RPMI 1640, MEM, DMEM, Ham F12, M199, and the like, and vascular endothelial growth factor, heparin, antibiotics and the like may be added. The culture conditions for vascular endothelial cells are not particularly limited, and it is preferable to perform the culture under 37 ° C., 5% CO ₂ , 95% air, saturated water vapor conditions for 2 to 4 days.

The therapeutic agent for rheumatism of the present invention targets neovascular endothelial cells in the joint synovial region during rheumatism and desirably does not act on vascular endothelial cells of normal tissues. From such a viewpoint, it is more preferable to use vascular endothelial cells in a proliferating phase having an antigen specific for neovascular endothelial cells, that is, cultured vascular endothelial cells.
Preferable examples include cultured vascular endothelial cells immediately after the growth phase or confluence, or vascular endothelial cells isolated from tissues undergoing angiogenesis.

  Furthermore, vascular endothelial cells are preferably stimulated with TNF-α, lipopolysaccharide (LPS), vascular endothelial growth factor (VEGF), or the like. Of these, the use of TNF-α-stimulated cultured vascular endothelial cells is particularly preferred for enhancing the effect of treating rheumatism. For stimulation of cultured vascular endothelial cells with TNF-α, for example, cultured vascular endothelial cells may be cultured for 10 to 40 hours in the presence of 10 to 500 U / mL of TNF-α.

  The vascular endothelial cell-derived antigen may be the vascular endothelial cell itself, or a vascular endothelial cell lysate or a vascular endothelial cell membrane vesicle. The cell disruption solution and membrane vesicle are more preferable because they are easy to prepare and have high stimulation efficiency for dendritic cells.

The cell lysate can be prepared, for example, by centrifuging (400 rpm, 10 minutes) at a low speed after repeating freezing (−160 ° C.) and thawing (37 ° C.) 4 to 6 times. Membrane vesicles, for example, are those obtained by adding 100 mM paraformaldehyde, 2 mM dithiothreitol, 1 mM CaCl ₂ , 0.5 mM MgCl ₂ to DMEM at 37 ° C., 5% CO ₂ , 95% air, saturated water vapor conditions. It can be processed overnight and then centrifuged at 150 xg for 5 minutes, and then the supernatant can be prepared by centrifugation at 30,000 xg for 30 minutes at 4 ° C.

  Among the active ingredients of the therapeutic agent for rheumatism of the present invention, (A) dendritic cells stimulated with an antigen derived from vascular endothelial cells are obtained by pulsing (stimulating) dendritic cells with an antigen derived from vascular endothelial cells. As the dendritic cells for pulsing the antigen, it is preferable to use human-derived dendritic cells, particularly dendritic cells derived from the patients themselves to be administered, or human-derived dendritic cells compatible with the patient and MHC. Dendritic cells can be obtained by inducing differentiation from bone marrow cells, umbilical cord-derived cells, peripheral blood mononuclear cells and the like. In order to induce differentiation of dendritic cells from peripheral blood mononuclear cells, for example, the cells are cultured for 5 to 10 days in a medium containing GM-CSF (10-100 ng / mL) and IL-4 (50-500 IU / mL). Is desirable.

Stimulation of dendritic cells with the vascular endothelial cell-derived antigen is performed, for example, by adding the vascular endothelium-derived antigen to a suspension of dendritic cells and incubating for 4 to 24 hours. In addition, it is preferable to use cationic liposomes or the like together so that antigen stimulation can be performed more efficiently. Furthermore, vascular endothelial cell antigens may be presented to dendritic cells by a cell fusion method between dendritic cells and vascular endothelial cells. The added amount of the antigen is preferably 0.01 to 100 μg / mL as a protein concentration per 1 × 10 ⁶ dendritic cells.

  The dendritic cells thus obtained present vascular endothelial cell-specific antigens of rheumatoid joint synovial tissue in vivo, and cytotoxic T cells (CTL) to the rheumatoid joint synovial tissue vascular endothelial cells are induced, thereby causing rheumatism. Vascular endothelial cells in the joint synovial tissue are attacked, blood vessels in the rheumatoid joint synovial tissue break down, inhibit the growth of the synovial tissue, and thereby suppress the increase in the synovial tissue, resulting in the healing of the rheumatic lesion Therefore, it is useful as a therapeutic agent for rheumatism.

Since the obtained cell suspension containing dendritic cells is administered to humans as a therapeutic agent for rheumatism, it is preferable to eliminate cell proliferation. In order to use it more safely, it can be treated under conditions such as heat treatment, radiation treatment, mitomycin C treatment, etc., while leaving the function as a therapeutic agent for rheumatoid arthritis, while the protein of the rheumatoid joint synovial tissue cells is denatured. .
For example, when X-ray irradiation is used, a flask containing dendritic cells is placed under the tube of the X-ray irradiator and irradiated with a total radiation dose of 1,000 to 3,300 Rad. In the mitomycin C treatment method, for example, dendritic cells are suspended at a density of 1 to 3 × 10 ⁷ cells / mL, added at a ratio of 25 to 50 μg of mitomycin C per 1 mL of cell suspension, and incubated at 37 ° C. and 30 to 60 ° C. Incubate for minutes. The cell treatment method using heat can be prepared, for example, by subjecting a centrifuge tube containing a cell suspension adjusted to a live cell concentration of 1 × 10 ⁷ cells / mL to a heat treatment at 50 to 65 ° C. for 20 minutes.

  The dendritic cells of the present invention can be used by the patient himself, but can be administered to many patients of the same type who are MHC compatible by the development of bone marrow bank and cord blood bank.

  Among the active ingredients of the therapeutic agent for rheumatism of the present invention, (B) the vascular endothelial cell-derived antigen and the pharmaceutically acceptable adjuvant may be a single preparation containing the antigen and the adjuvant, or the antigen and the adjuvant are separated. And may be used as a mixture at the time of administration.

  The pharmaceutically acceptable adjuvant used in combination with the antigen may be any adjuvant that can be used in humans, but is of a sedimenting type such as an aluminum hydroxide gel used as an adjuvant for pathogenic microorganism vaccine preparations, There are emulsion type using olive oil, polysaccharides such as pullulan, and montanide adjuvant. The ratio of the antigen to the adjuvant varies depending on the type of the adjuvant. When aluminum hydroxide gel is used as the adjuvant, the amount of aluminum hydroxide contained in the gel is defined as the amount of adjuvant. ) = 1: 100 to 100: 1, particularly 1:50 to 50: 1.

  In addition to the above-mentioned active ingredients, the therapeutic agent for rheumatism of the present invention is used in combination with a non-steroidal anti-inflammatory drug, a corticosteroid, a disease-modifying anti-rheumatic drug, methotrexate, a metal agent, a biological agent such as an antibody drug, etc. Also good.

  In addition, the therapeutic agent for rheumatism of the present invention is (A) a preparation that induces mucosal immunity such as subcutaneous, intradermal, intramuscular injection, oral, nasal mucosa, etc. in the case of an antigen derived from vascular endothelial cells and an adjuvant. Can be administered as In addition, (B) dendritic cells can be administered as an injection such as subcutaneous, intradermal, intramuscular: intravenous. In preparing these preparations, pharmaceutically acceptable carriers such as solubilizers, buffers, isotonic agents, preservatives, suspending agents, emulsifiers and the like can be blended.

  The dose of the therapeutic agent for rheumatism of the present invention varies depending on the patient's age, weight, sex, progression of rheumatism, symptoms, etc., and cannot be determined in general, but the amount of vascular endothelial cells or dendritic cells is 1 μg per dose. About 100 mg is preferable.

  The present invention will be described in more detail with reference to the following examples. However, these examples do not limit the scope of the present invention.

Example 1
A. Experimental Method (1) Preparation of Type II Collagen Arthritis Mice Type II collagen induced arthritis mice widely used as rheumatoid arthritis models were prepared and used. In other words, type II collagen solution and Freund's complete adjuvant are mixed in equal amounts with a glass syringe to make an emulsion, immunized into the ridge skin of DBA / 1J mice, and type II collagen solution 3 weeks after the initial sensitization. A booster immunization was performed intraperitoneally.

(2) Vascular endothelial cells Mouse hepatic sinusoidal endothelial cells (HSE) and human umbilical vein endothelial cells (HUVEC) were used as vascular endothelial cells. (These vascular endothelial cells are not vascular endothelial cells in the synovial region, but since these cells actively proliferate under in vitro conditions, vascular endothelial cells of new blood vessels actively proliferating in vivo. It was thought to be similar in nature and used as a vascular endothelial cell model for new blood vessels.)

(3) Preparation of antigen A 2.5% (v / v) 1-butanol / PBS solution was added to cultured vascular endothelial cells, and the extracted solution was dialyzed and lyophilized to obtain an antigen.

(4) Immunization with adjuvant Lyophilized antigen was dissolved in physiological saline, and an equal volume of adjuvant (Titer Max ^R Gold) was mixed and injected subcutaneously into the back of the neck of a rheumatoid model mouse. Immunization was performed one week before the initial sensitization of type II collagen, and antigen immunization with an adjuvant was performed again three weeks later.

(5) Preparation of mouse bone marrow-derived dendritic cells (DC) Bone marrow tissue is removed from the femur and tibia of DBA / 1J mice, and GM-CSF (granulocyte macrophage colony-stimulating factor) What was differentiated into DC (dendritic cells) by culturing in the containing medium was used.

(6) Antigen pulse to DC and DC immunization Each freeze-dried antigen was pulsed to DC using lipofectin ^™ . This DC was immunized into the back of the mouse. Immunization with DC was performed one week after the initial sensitization of type II collagen, and another week later, DC was immunized.

(7) Scoring of Rheumatoid Arthritis Score The arthritis score for the limbs of rheumatoid arthritis model mice was scored daily according to a conventional method, and rheumatoid arthritis was evaluated by the total score. The score was scored in five stages from 0 to 4 as follows.

0: No change 1: Redness of only one finger joint 2: Redness of two or more finger joints or large joints such as wrists and ankles 3: Redness of swelling of one hand and the whole leg 4: Maximum swelling of an entire hand or foot

The scoring evaluation ended on the 50th day.
The initial sensitization of type II collagen was defined as day 0, and the scores of mouse limbs were scored according to Wood et al. And measured until day 50. One week prior to type II collagen sensitization, the antigen was immunized with an adjuvant. The second immunization was one week before the second sensitization of type II collagen. The total score of each mouse limb was taken as the score of that individual, and each group was shown as an average score (n = 5).

B. Experimental Results (1) FIG. 1 shows the daily changes in the rheumatoid arthritis score when immunized with vascular endothelial cell antigen using an adjuvant. In the control group (no adjuvant administered) and the adjuvant only group (no vascular endothelial cell antigen), swelling redness due to rheumatoid arthritis began to appear on the 20th day after the initial sensitization of type II collagen, and the adjuvant on the 33rd day. Swelling redness appeared in all mice in the only group, and the arthritis score increased with the passage of days. The control group showed almost the same results, and the effect of suppressing or promoting rheumatoid arthritis by administration of the adjuvant itself was not recognized. On the other hand, in both groups using HSE and HUVEC as antigens, swelling and redness of rheumatoid arthritis did not appear in almost all mice, and rheumatoid arthritis was remarkably suppressed.
Since the rheumatoid arthritis inhibitory effect was not recognized in the adjuvant only group, the suppression of rheumatoid arthritis in the HSE and HUVEC groups may be due to immunization with vascular endothelial cells as an antigen, not the action of the adjuvant itself. It was suggested.

(2) FIG. 2 shows the daily change of the rheumatoid arthritis score when immunized with DC pulsed vascular endothelial cell antigen. From the 20th day after the initial sensitization of type II collagen in both the control group (no DC administration) and the DC only group (administration of the DC group not pulsed with antigen), swelling redness due to rheumatoid arthritis began to appear. On the day, swelling and redness appeared in all mice in the antigen-untreated group, and the rheumatoid arthritis score increased with the passage of days. The control group showed almost the same results, and the effect of suppressing or promoting rheumatoid arthritis by DC administration was not observed.
On the other hand, in both groups to which DCs pulsed with HSE and HUVEC were administered, swelling redness of rheumatoid arthritis did not appear in almost all mice, and rheumatoid arthritis was remarkably suppressed.
Since the rheumatoid arthritis inhibitory effect was not recognized in the DC only group, the suppression of rheumatoid arthritis in the HSE and HUVEC administration groups was not an anti-rheumatic effect due to an increase in nonspecific immune function by DC administration, but a new blood vessel It was inferred that the anti-rheumatic effect was attributed to immunity to neovascular endothelial cells caused by pulsing endothelial cells with antigen as an antigen and inhibiting angiogenesis at the joint synovial site.

(3) Observation of bone destruction by X-ray photograph As for the degree of rheumatoid arthritis, “swelling” of the appearance is important, but evaluation of the degree of joint bone destruction accompanied by dysfunction is also important. Therefore, radiographs were taken to observe the degree of bone destruction.
50 days after the start of the experiment, the mice were sacrificed, the limbs were cut, and formalin was fixed. Thereafter, radiographs were taken to observe the degree of bone destruction.
The results of adjuvant treatment are shown in FIG. The bones of mice that did not develop rheumatoid arthritis were dense and the joint structure was clear. On the other hand, in mice with rheumatoid arthritis, bone density decreased, bone destruction was observed, the joints were also unclear, and it was confirmed that the swelling had progressed to joint destruction at the bone level . In the vascular endothelial cell immunity group, as in the normal mouse group, no decrease in bone density or joint destruction was observed, and not only the appearance of joint swelling but also the evaluation of bone by X-rays, rheumatoid arthritis It was confirmed that there was almost no onset. In the vascular endothelial cell immunity group, a dense bone structure was observed as in the normal mouse group, and it was observed that the onset of rheumatoid arthritis was remarkably suppressed.

(4) Since the bone change due to the onset of rheumatoid arthritis is markedly evaluated at the heel site, FIG. 4 shows the bone change at the heel site. The bones of the buttocks of normal mice that did not develop rheumatoid arthritis were dense. On the other hand, in the wrinkles of mice that developed rheumatoid arthritis, a marked decrease in bone density was observed, confirming that rheumatoid arthritis had progressed to the fistula site. In the vascular endothelial cell immunity group, a dense bone structure was observed as in the normal mouse group, and it was observed that the onset of rheumatoid arthritis was remarkably suppressed even in the heel region.

Example 2
For the purpose of optimizing vascular endothelial cells that are antigens, that is, to reproduce the state closer to vascular endothelial cells in the synovial region of rheumatoid arthritis and to induce immunity against arthritic vascular endothelial cells, TNF-α stimulated vascular endothelial cells And their usefulness were studied.

(1) TNF-α-stimulated vascular endothelial cells Since TNF-α is involved in the exacerbation of rheumatism and TNF-α shows a high value at the joint site, blood vessels stimulated with TNF-α (100 U / mL, treated for 24 hours) Endothelial cells were used as a joint synovial vascular endothelial cell model.
Arthritis score was scored. FIG. 5 shows the result. The group using TNF-α-stimulated vascular endothelial cells as an antigen suppressed the onset of rheumatoid arthritis more markedly than the group using untreated vascular endothelial cells (HSE) as an antigen previously found.

Example 3
Whether this rheumatic effect is due to induction of immunity to vascular endothelial cells was evaluated by measuring anti-vascular endothelial cell antibody titer in mouse blood.

(1) Measurement of anti-vascular endothelial cell antibody titer 50 days after the start of the experiment, blood was collected from the mouse, and the anti-vascular endothelial cell antibody titer in the obtained serum was immobilized on vascular endothelial cell antigen (HSE or HUVEC). Measured by ELISA using the prepared plate.

  The results are shown in FIG. When immunized with an adjuvant, it was shown that the antibody titer against HSE (mouse vascular endothelial cells) was markedly increased when immunized with HSE as an antigen, so that immunity against vascular endothelial cells was induced. It became clear that. Furthermore, even when immunized with HUVEC (human vascular endothelial cells) as an antigen, an increase in antibody titer against HSE was observed. Further, HUVEC was immobilized and the same study was conducted. In both cases of immunization using HSE or HUVEC as an antigen, a significant increase in anti-vascular endothelial cell antibody titer was observed. It was inferred that there was a common antigen, immunity against the antigen was induced, neovascular endothelial cells were targeted, neovascular failure occurred, and antirheumatic effects were exhibited.

Example 4
In type II collagen-induced rheumatoid arthritis, the anti-type II collagen antibody titer in blood is high, and this antibody contributes to the destruction of synovial tissue. Steroidal drugs such as adrenal hormones, which are one of rheumatoid therapeutic drugs, exhibit antirheumatic effects with immunosuppression as the mechanism of action. Therefore, when the anti-rheumatic effect is recognized by the steroid drug, a decrease in the anti-type II collagen antibody titer in the blood is observed. Therefore, in order to elucidate the mechanism of action of this immunotherapy targeting neovascular endothelial cells, the blood type II collagen antibody titer was measured.

(1) Measurement of anti-type II collagen antibody titer 50 days after the start of the experiment, blood was collected from the mouse, and the obtained serum was used to measure the anti-type II collagen antibody titer by ELISA.
The results of measuring the blood anti-type II collagen antibody titer by ELISA are shown in FIG. The group immunized with HSE or HUVEC as an antigen using an adjuvant or DC produced an anti-type II collagen antibody that was not different from the antigen-untreated group. As a result, it was suggested that the mechanism of action of this immunotherapy is different from nonspecific suppression of autoimmunity involved in arthritis development. From these results, it was speculated that this therapy is targeted to neovascular endothelial cells.

Example 5
We examined the possible side effects of this therapy. The vascular endothelial cells used in this study are vascular endothelial cells of normal tissue, and are used as a neovascular model by preparing an antigen in the cell growth phase under culture conditions. Therefore, if normal tissue vascular endothelial cells are recognized as antigens regardless of the growth phase of the cells, and normal tissue vascular endothelial cells in immunized animals are destroyed, tissue dysfunction is caused and extremely serious side effects are caused. The possibility of developing it cannot be denied. Therefore, since HSE is a mouse liver-derived vascular endothelial cell-derived cell, whether or not hepatic injury has occurred due to the breakdown of liver blood vessels was evaluated from the biochemical values of GPT and GOT in mouse blood.

Blood biochemical test 50 days after the start of the experiment, blood was collected from the mouse, and blood GPT (ALT) and GOT (AST) activities were measured. The results are shown in Table 1.
The values of GPT and GOT activity in the antigen-administered groups (HSE, HUVEC) showed no difference from the untreated group and the antigen-untreated group when adjuvant was used and when DC was used, and liver damage was observed. There wasn't.

  In Example 1 (1), since the arthritis suppressing effect was not recognized in the adjuvant only group, the suppression of arthritis in the HSE and HUVEC groups was not an action of the adjuvant itself, but immunization using vascular endothelial cells as an antigen. It was suggested that

  In Example 1 (2), since no arthritis suppressing effect was observed in the group of only DC (dendritic cell: dendritic cells), the suppression of arthritis in the HSE and HUVEC administration groups was performed by non-specific immunization by DC administration. Anti-rheumatic effect due to inhibition of angiogenesis in joint synovial site caused by immunity to neovascular endothelial cells caused by pulsing neovascular endothelial cells to DC as antigen instead of antirheumatic effect due to increased function It was inferred that this was an effect.

  In Example 3, a common antigen is present in neovascular endothelial cells of any species, immunity against the antigen is induced, the neovascular endothelial cells are targeted, neovascular disruption occurs, and an anti-rheumatic effect is exhibited. It was guessed.

  In Example 4, it was suggested that the mechanism of action of this immunotherapy is different from nonspecific suppression of autoimmunity involved in arthritis development. From these results, it was speculated that this therapy is targeted to neovascular endothelial cells.

We examined the possible side effects of this therapy. The vascular endothelial cells used in this study are vascular endothelial cells of normal tissue, and are used as a neovascular model by preparing an antigen in the cell growth phase under culture conditions. Therefore, if normal tissue vascular endothelial cells are recognized as antigens regardless of the growth phase of the cells, and normal tissue vascular endothelial cells in immunized animals are destroyed, tissue dysfunction is caused and extremely serious side effects are caused. The possibility of developing it cannot be denied. Therefore, since HSE is a mouse liver-derived vascular endothelial cell-derived cell, whether or not hepatic injury has occurred due to the breakdown of liver blood vessels was evaluated from the biochemical values of GPT and GOT in mouse blood (implementation) Example 5).
The anti-rheumatic effect is recognized by this therapy (Examples 1 and 2), and Examples 3 and 4 suggest that immunity to new vascular endothelial cells is induced, but liver damage is From the results of Example 5 that were not observed, it is inferred that the present immunotherapy is in an immune-tolerant state without inducing immunity to vascular endothelial cells in a stationary state in the growth arrest phase.
This immunotherapy targeting only new blood vessels was found to be a safe treatment with few side effects without causing dysfunction due to vascular endothelium failure in normal tissues.
Therefore, the usefulness of anti-rheumatoid arthritis immunotherapy using neovascular endothelial cells as an antigen was shown here.

It is a figure which shows the inhibitory effect of rheumatoid arthritis by immunizing with a vascular endothelial cell antigen with an adjuvant. It is a figure which shows the inhibitory effect of rheumatoid arthritis by immunizing the dendritic cell pulsed with the vascular endothelial cell antigen. It is a limb X-ray photograph of a mouse with rheumatoid arthritis immunized with an angiogenic endothelial cell antigen together with an adjuvant. It is an extremity X-ray photograph (anther part) of a mouse with rheumatoid arthritis immunized with an angiogenic endothelial cell antigen together with an adjuvant. It is a figure which shows the arthritis score by the immunotherapy which used the vascular endothelial cell stimulated by TNF- (alpha) as an antigen. It is a figure which shows the measurement result of the anti-vascular endothelial cell antibody titer in the immunotherapy using an adjuvant. It is a figure which shows the mouse | mouth blood anti-type II collagen antibody titer in the immunotherapy using adjuvant and DC.

Claims (9)

A therapeutic agent for rheumatism comprising (A) dendritic cells stimulated with an antigen derived from vascular endothelial cells, or (B) an antigen derived from vascular endothelial cells and a pharmaceutically acceptable adjuvant.   The therapeutic agent for rheumatism according to claim 1, wherein the antigen derived from vascular endothelial cells is a vascular endothelial cell, a cell lysate thereof or a cell membrane vesicle thereof.   The therapeutic agent for rheumatism according to claim 1 or 2, wherein the vascular endothelial cell is a cultured vascular endothelial cell. Use of (A) dendritic cells stimulated with an antigen derived from vascular endothelial cells, or (B) an antigen derived from vascular endothelial cells and a pharmaceutically acceptable adjuvant for the production of a therapeutic agent for rheumatism.   The use according to claim 4, wherein the vascular endothelial cell-derived antigen is a vascular endothelial cell, a cell lysate thereof or a cell membrane vesicle thereof.   The use according to claim 4 or 5, wherein the vascular endothelial cell is a cultured vascular endothelial cell. A method for treating rheumatism, comprising administering (A) dendritic cells stimulated with an antigen derived from vascular endothelial cells, or (B) an antigen derived from vascular endothelial cells and a pharmaceutically acceptable adjuvant.   The method according to claim 7, wherein the antigen derived from vascular endothelial cells is a vascular endothelial cell, a cell lysate thereof or a cell membrane vesicle thereof.   The method according to claim 1 or 8, wherein the vascular endothelial cells are cultured vascular endothelial cells.