JP5224325B2 - B cell malignant lymphoma treatment - Google Patents

Description

  The present invention relates to a drug for treating B cell malignant lymphoma.

  Although malignant tumors occupy the top causes of death in various countries, especially developed countries, the current cure has not been established yet. Methods for treating malignant tumors mainly include chemotherapy, such as surgery, radiation therapy, and administration of antitumor drugs, each having its advantages and disadvantages.

  Among malignant tumors, malignant lymphoma occurs in the lymphatic system, and unlike the cancer derived from epithelial cells, the lymphatic system itself is a systemic tissue and cannot be removed by surgical operation. Therefore, radiation therapy and chemotherapy are generally employed as treatment means.

  Since chemotherapy can be applied to the whole body, it is effective for progressive tumors with microtumor tissue or metastatic potential. Since tumor cells are originally cells that proliferate in a disorderly manner against normal control, radiation and antitumor drugs have a therapeutic effect on tumors, but they also have the drawback of damaging normal cells There is. Therefore, the advent of better antitumor drugs that have excellent therapeutic effects on tumors and few side effects is expected.

  However, due to the nature of malignant lymphoma occurring throughout the body, it is not possible to prove that the tumor cells have completely disappeared. On the contrary, it is considered difficult to treat because there are many cases of recurrence after treatment.

  Malignant lymphomas are classified into various categories, and the incidence varies depending on race, country and region. For example, lymphocytes include B cells, T cells, and NK cells. In Japan, 70 to 80% are B cell malignant lymphomas, 20 to 30% are T cell malignant lymphomas, and NK cell malignant lymphomas are less than 5%. There is no data. Thus, since B cell malignant lymphoma may occur at a high ratio among malignant lymphomas, an effective therapeutic means is required.

  By the way, HMGB1 (High Mobility Group box 1) is a protein having the same amino acid sequence of 95% or more from rodents to humans. Although this HMGB1 is also present in normal cells, the blood concentration is increased by stimulation with LPS (lipopolysaccharide) which is an endotoxin released in sepsis (systemic inflammatory response syndrome), resulting in final tissue damage . Therefore, in the technique described in Patent Document 1, an HMGB1 antagonist is administered to treat a symptom characterized by activation of the inflammatory cytokine cascade, and lymphoma is also exemplified as a treatment target. However, although the patent document 1 discloses an experimental example in which the mortality rate of a septic model mouse administered with a lethal dose of LPS is decreased by administration of an anti-HMGB1 antibody, the effect of the anti-HMGB1 antibody on other diseases is demonstrated. It has not been.

  Patent Document 2 discloses a composition containing an HMGB1 antibody and the like as a composition for treating a side effect induced by necrotic tissue. However, examples of such side effects include activation of nearby living cells, recruitment and activation of bone marrow cells, loss of endothelial barrier function, and edema, but no B cell malignant lymphoma is described.

In addition, the present inventors have continued research on anti-HMGB1 antibodies and have found the anti-HMGB1 antibody's inhibitory effect on cerebral infarction and cerebral vasospasm as described in Patent Documents 3 and 4.
Japanese translation of PCT publication No. 2003-520863 JP 2005-537253 A JP 2007-119347 A Japanese Patent No. 3882090

  As described above, antitumor drugs can generally damage not only tumor cells but also normal cells. In addition, since B cell malignant lymphoma has a relatively high incidence rate among malignant lymphomas, a better therapeutic means is required.

  Therefore, the problem to be solved by the present invention is to provide a drug having a therapeutic effect on B-cell malignant lymphoma and having few side effects.

  In order to solve the above problems, the present inventors have made various studies on drugs effective for the treatment of B-cell malignant lymphoma. As a result, it was found that the anti-HMGB1 monoclonal antibody has a very excellent effect, and the present invention was completed.

  The therapeutic agent for B-cell malignant lymphoma of the present invention comprises an anti-HMGB1 monoclonal antibody as an active ingredient.

  As a dose of the anti-HMGB1 monoclonal antibody, it is considered that 0.2 to 5 mg / (kg body weight) per dose is suitable from the experimental results described later. In addition, since the main component of the therapeutic agent for B cell malignant lymphoma of the present invention is an antibody, intravenous administration is suitable as an administration form.

  The therapeutic agent of the present invention can effectively suppress B cell malignant lymphoma having a relatively high incidence among malignant lymphomas. In addition, considering the currently used antibody drugs, it is very unlikely that serious side effects will occur. Therefore, the therapeutic agent for B-cell malignant lymphoma of the present invention is extremely useful as an agent that can effectively treat B-cell malignant lymphoma for which better treatment means are desired without side effects.

  The B cell malignant lymphoma of the present invention contains an anti-HMGB1 monoclonal antibody as an active ingredient.

  B-cell malignant lymphoma is a type of non-Hodgkin's malignant lymphoma. Non-Hodgkin's malignant lymphoma includes B-cell malignant lymphoma and T-cell malignant lymphoma, but the incidence of B-cell malignant lymphoma may be overwhelmingly higher than T-cell malignant lymphoma depending on the race and region. The symptom is swollen lymph nodes, but it is generally painless and worsens without subjective symptoms. Since B-cell malignant lymphoma is systemic, it is generally treated with multi-drug chemotherapy, depending on the age and severity of the patient. Therefore, since the burden on patients due to side effects of anticancer drugs is large, anticancer drugs with fewer side effects have been demanded for B-cell malignant lymphoma.

  The present inventors first found that HMGB1 increases the proliferation of B cell malignant lymphoma in a concentration-dependent manner, and further confirmed that the anti-HMGB1 monoclonal antibody significantly suppresses the proliferation of B cell malignant lymphoma. A similar experiment was conducted for T-cell malignant lymphoma, but it sometimes proliferated by anti-HMGB1 antibody. Therefore, according to the knowledge of the present inventors at the present stage, it is considered that the effect of suppressing cell proliferation by the anti-HMGB1 antibody is specific to B cell malignant lymphoma.

  The anti-HMGB1 monoclonal antibody selectively acts on HMGB1, which is considered to be involved in inflammatory reactions, and the mechanism of action is not clear, but suppresses the growth of B cell malignant lymphoma. On the other hand, it basically does not act on other chemical mediators. Therefore, it is thought that there is no possibility that a side effect will occur or very little.

  The anti-HMGB1 monoclonal antibody may be prepared according to a conventional method. For example, a mouse, a rat, or the like is immunized using commercially available HMGB1, and the antibody-producing cells, spleen cells, and myeloma cells are fused to obtain hybridomas. The hybridoma is cloned, and a clone producing an antibody that specifically reacts with HMGB1 is screened. This clone may be cultured and the secreted monoclonal antibody may be purified.

  The type of anti-HMGB1 monoclonal antibody used in the present invention is not particularly limited. For example, a human type antibody or a fully human antibody can be used.

  The dosage form of the therapeutic agent for B-cell malignant lymphoma according to the present invention is not particularly limited, but considering that the anti-HMGB1 monoclonal antibody as an active ingredient is a peptide, it is intended to be administered as an injection and is used as a solution or emulsion. A liquid preparation such as a preparation is preferred.

  As a solvent for the liquid preparation, an isotonic solution of plasma such as physiological saline adjusted with pH or aqueous glucose solution can be used. In addition, when the antibody is lyophilized with salts or the like, pure water, distilled water, sterilized water, or the like can be used. The concentration may be that of a normal antibody preparation, generally about 0.1 to 1 mg / mL, and about 0.02 to 0.2 mg / mL for infusion. However, the osmotic pressure of the injection must be equivalent to that of plasma.

  The therapeutic agent for B-cell malignant lymphoma of the present invention is used alone or in combination with other antitumor drugs or therapeutic means. When used in combination with other anti-tumor drugs, it is considered that the usual amount of use of other anti-tumor drugs can be suppressed, and as a result, its side effects can be reduced.

  The administration frequency and dose of the therapeutic agent for B-cell malignant lymphoma according to the present invention may be appropriately adjusted depending on the severity and age of the patient. As shown in Examples described later, a sufficient B cell malignant lymphoma growth inhibitory effect is observed even in a medium having an anti-HMGB1 monoclonal antibody concentration of 1 μg / mL. Therefore, the concentration of the preparation to be administered and the dose of the anti-HMGB1 monoclonal antibody may be appropriately adjusted. For example, 0.2 to 5 mg / (kg body weight) can be administered at one time.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. It is also possible to implement, and they are all included in the technical scope of the present invention.

Example 1 Preparation of anti-HMGB1 monoclonal antibody (a) Immunization of rat Commercially available mixture of bovine thymus-derived HMGB1 and HMGB2 (manufactured by Wako Pure Chemical Industries, Ltd., code number: 080-070741) 1 mg / mL in a 2 mL glass syringe The mixture was gradually mixed with an equal volume of Freund's complete adjuvant taken in a separate 2 mL glass syringe through a connecting tube to obtain an emulsion. The resulting emulsion was administered by injection into the hind footpads of rats anesthetized with sevoflurane in an amount of 0.1 mL, for a total of 0.2 mL. Two weeks later, test blood was collected from the jugular vein, and an increase in antibody titer was confirmed. The swollen iliac lymph nodes were then aseptically removed 5 weeks after the injection administration. Approximately 6 × 10 ⁷ cells could be recovered from the two lymph nodes obtained.

(B) Cell fusion and cloning The iliac lymph node cells and mouse myeloma SP2 / O-Ag14 (SP2) cells were fused using polyethylene glycol, and the resulting fused cells were seeded in a 96-well microplate. One week later, an initial ELISA screening was performed and positive wells were secondary screened by Western blot. Positive well cells are transferred to a 24-well microplate, and the cells are grown to a nearly confluent state (about 2 × 10 ⁵ cells). Then, 0.5 mL of freezing medium (GIT medium containing 10% fetal bovine serum and dimethyl And 10% sulfoxide added) and stored frozen in liquid nitrogen. The cryopreserved cells were thawed and then cloned in a 96-well microplate.

(C) Purification of antibody The positive cells were cultured in a large amount for 2 weeks using a rotary culture apparatus (manufactured by Vivascience) to obtain an antibody solution having a concentration of 2 to 3 mg / mL. This antibody solution was mixed with an affinity gel (MEP-HyperCel, manufactured by Invitrogen) under neutral pH to specifically bind the anti-HMGB1 antibody to the gel. The antibody specifically bound to the gel was eluted with glycine-hydrochloric acid buffer (pH 4). The eluate was concentrated by an ultrafiltration device and further purified by a Sepharose CL6B gel filtration column (diameter 2 cm × length 97 cm).

  The resulting monoclonal antibody is an antibody that specifically recognizes 208EEEDDDDE215 (E indicates glutamic acid and D indicates aspartic acid), which is the C-terminal sequence of HMGB1 protein, as an epitope. For example, there is HMGB2 as a protein similar to HMGB1, but since HMGB2 does not have a DDDDE sequence of 211 or less, the monoclonal antibody according to the present invention does not bind to HMGB2 and specifically recognizes and binds only to HMGB1. can do.

Example 2 Cell Proliferation Experiment with HMGB1 Separately prepared human HMGB1 was dissolved in a liquid medium (manufactured by Nissui, product name “RPMI 1640”) at a concentration of 1 × 10 ⁻¹⁰ to 1 × 10 ⁻⁶ g / mL. Moreover, the culture medium which does not add HMGB1 was also prepared for the comparison. These culture media (1 mL) were added to a petri dish having a diameter of 2 cm, and B18 malignant lymphoma strains FL18 and FL218 (obtained from Prof. Hitoshi Ono of Ichinaikai Takeda General Hospital) were added at a rate of about 200,000 cells / mL, respectively. FL18 is a human B-cell malignant lymphoma strain, and FL218 is FL18 deficient in the Bcl-2 gene having an inhibitory effect on apoptosis. These B cell malignant lymphoma lines were cultured at 37 ° C. for 48 hours, and then the cell concentration in each medium was measured by observing with a microscope. The result of FL18 is shown in FIG. 1 (1), and the result of FL218 is shown in FIG. 1 (2). Note that “*” in FIG. 1 indicates that there is a significant difference at p <0.05 when the obtained result is tested in Dunnet's test, and “**” is p <0.01. The case where there is a significant difference is shown.

As shown in FIG. 1, it was found that HMGB1 promotes the growth of B cell malignant lymphoma B in a concentration-dependent manner, and significantly increases the growth rate particularly at a concentration of 1 × 10 ⁻⁷ to 1 × 10 ⁻⁶ g / mL. .

Example 3 Cell Proliferation Inhibition Experiment with Anti-HMGB1 Monoclonal Antibody Two types of B cells were prepared in the same manner as in Example 2 except that the anti-HMGB1 monoclonal antibody obtained in Example 1 was dissolved in a liquid medium at a concentration of 1 μg / mL. The malignant lymphoma line was cultured at 37 ° C. for 48 hours. The concentration of cells in each medium was measured in the same manner as in Example 2 immediately after the start of culture and at 2 hours, 6 hours, 18 hours, 24 hours and 48 hours after the start of culture. The result of FL18 is shown in FIG. 2 (1), and the result of FL218 is shown in FIG. 2 (2).

  As shown in FIG. 2, the anti-HMGB1 monoclonal antibody suppresses the growth of B cell malignant lymphoma over time. Therefore, it was demonstrated that the anti-HMGB1 monoclonal antibody according to the present invention has a therapeutic effect on B cell malignant lymphoma.

It is a graph which shows the result of the proliferation experiment of B cell malignant lymphoma by HMGB1. (1) shows the result of FL18, and (2) shows the result of FL218. It is a graph which shows the result of the growth-inhibition experiment of B cell malignant lymphoma by anti- HMGB1 monoclonal antibody. (1) shows the result of FL18, and (2) shows the result of FL218.

Claims (5)

  A therapeutic agent for B cell malignant lymphoma comprising an anti-HMGB1 monoclonal antibody as an active ingredient. The therapeutic agent for B cell malignant lymphoma according to claim 1, wherein the anti-HMGB1 monoclonal antibody has a C-terminal sequence of HMGB1 protein as an epitope. The therapeutic agent for B cell malignant lymphoma according to claim 1, wherein the anti-HMGB1 monoclonal antibody has 208EEEDDDDE215, which is the C-terminal sequence of HMGB1 protein, as an epitope. The therapeutic agent for B cell malignant lymphoma according to any one of claims 1 to 3, wherein the anti-HMGB1 monoclonal antibody is administered at a dose of 0.2 to 5 mg / (kg body weight) once. The therapeutic agent for B cell malignant lymphoma according to any one of claims 1 to 4, which is administered intravenously.

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