JP2008105953A - Cytotoxicity inhibitor for b cells - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cytotoxicity inhibitor for B cells capable of inhibiting toxy activity to the B cells by a mucine, and recovering antibody producing ability. <P>SOLUTION: The cytotoxicity inhibitor for B cells includes an anti-mucine antibody as an active constituent. The anti-mucine antibody reduces the cytotoxicity of the mucine to the B cells by specifically bonding to the mucine in a body (in blood) so as to recover or improve the antibody producing ability of the B cells. Hence, it has a potential for using for prevention and treatment for various disorders (immune disorder, autoimmune diseases and the like) caused by or believed to be caused by killing or function reduction of the B cells. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a B cell injury inhibitor. More specifically, the present invention relates to a B cell injury inhibitor that can suppress / recover injury of B cells that are immune cells.

Mucin is a glycoprotein having an infinite number of sugar chains bound to a polypeptide (core protein) through an O-glycoside bond. Mucins cover the lumen of the digestive organs such as the trachea, gastrointestinal tract, and gonads, and have cytoprotective effects, as well as cell-cell interactions (interactions with leukocytes, bacteria, and viruses) due to their sugar chain structures. ing.
By the way, mucin that cannot be detected normally is detected in the blood of epithelial cancer patients, and it is currently known that at least nine types of epithelial mucin (mucin derived from epithelial cancer cells) are present ( Non-patent document 1). In epithelial cancer patients, mucin binds to B cell siglec 2 (CD22), suppresses signal transmission of B cell activation signals, and CD21 and CD1d positive cells, which are subpopulations of splenic B cells, It is specifically reduced, resulting in the failure to form a splenic marginal zone composed of the cells. As a result, in cancer-bearing patients, it has been known that the ability to produce antibodies against T cell-independent antigen, which is a function of B cells, is significantly reduced (see Non-Patent Document 2).
Glycobiology vol.10, No5, pp.439-449 (2000) Bulletin of the Research Institute for Advanced Science and Technology, Kyoto Sangyo University (published July 2004)

As described above, in patients with epithelial cancer, mucin reduces the ability to produce antibodies in B cells, so that there is a problem that immunity of cancer-bearing patients tends to decrease.
The present inventor has made various studies in order to solve such problems, and in order to enhance immunity of cancer-bearing patients, it is important to suppress / recover B cell damage. It was found that B cell injury can be suppressed by administering.
The present invention is based on such findings, and provides a B cell injury inhibitor capable of enhancing the immunity of a patient whose function is reduced due to injury to B cells such as cancer-bearing patients. To do.

  The present invention made in order to solve the above-mentioned problems is a B cell injury inhibitor containing an anti-mucin antibody as an active ingredient, and the anti-mucin antibody is preferably an antibody against epithelial mucin, and further, a sugar chain of mucin An antibody that recognizes the portion, more preferably a monoclonal antibody is used.

  According to the B cell injury inhibitor of the present invention, the anti-mucin antibody can specifically bind to the mucin in the body (in the blood) to reduce the mucin's toxicity to the B cell, and the antibody producing ability of the B cell. Recovery and improvement.

As described above, the present invention is a B cell injury inhibitor containing an anti-mucin antibody as an active ingredient.
The anti-mucin antibody used in the present invention means an antibody that can specifically bind to various mucins (particularly epithelial mucin), and the antibody may be a polyclonal antibody, a monoclonal antibody, or a mixture thereof. Methods for preparing antibodies are well known to those skilled in the art and can be prepared according to conventional methods.
Moreover, it is preferable to use a monoclonal antibody from the point which can acquire a uniform antibody stably. Furthermore, the epitope may be a core protein portion, but an anti-mucin antibody that recognizes a sugar chain region is preferable from the viewpoint of reactivity.

  The immunogen used for preparing the anti-mucin antibody is not particularly limited as long as it is an immunogen capable of producing an anti-mucin antibody (referred to as a mucin antibody-producing immunogen). Cancer cells (for example, human intestinal cancer cell LS180 and the like).

  More specifically, the anti-mucin polyclonal antibody can be prepared from the antiserum of immunized animals (for example, horses, cows, sheep, goats, rabbits, guinea pigs, rats, mice, etc.), for example, production of mucin antibodies containing an adjuvant The immunogen is administered subcutaneously to the immunized animal, and the administration is repeated at appropriate intervals and times (for example, about 4 to 6 times in 1 to 2 weeks), whole blood is collected after the final immunization, and antiserum is administered. To separate. Subsequently, the purified anti-mucin polyclonal antibody can be obtained by purifying the antiserum with a column using a carrier in which mucin is bound to BrCN-activated Sepharose or the like.

  The anti-mucin monoclonal antibody used in the present invention is a sensitized cell of an immunized animal (eg, rat, mouse, chicken, horse, cow, sheep, goat, rabbit, guinea pig, etc.) immunized with a mucin antibody-producing immunogen. A hybridoma is produced by fusing a proliferative cell such as a spleen cell or lymphocyte with a cell fusion technique to produce a monoclonal antibody that specifically reacts with mucin. Can be obtained by culturing the hybridoma in an appropriate medium or administering it into the peritoneal cavity of an animal to produce the antibody in ascites and collecting the produced monoclonal antibody.

The method for preparing the above anti-mucin antibody has already been published. For example, Biochemistry, Vol. 66, No. 1387-1400 (1994); Bulletin of Research Institute for Land Use, Kyoto Sangyo University, No. 10, No. 46-64 (1990); JP-A-3-280894; JP-A-6-172219; JP-A-7-101997, etc., which can be referred to.
The anti-mucin antibody is already used and sold as a clinical test reagent for examining a tumor marker (mucin), and the anti-mucin antibody used therein can also be used.

  The administration site / route to the patient of the B cell injury inhibitor of the present invention is not particularly limited as long as it is a site / route that can bind to mucin in vivo and express its action, but is generally administered intravenously. , Intraperitoneal administration, intraarterial administration, subcutaneous administration, intradermal administration, and the like, and intravenous administration is preferred. Furthermore, the administration route through a device embedded in the body is also mentioned. Specifically, a method of continuously and gradually administering to a patient using an osmotic pump or the like, or a sustained-release preparation (for example, a mini-pellet preparation) ) Is embedded in the patient.

  As a preparation form, various preparation forms (for example, a liquid agent etc.) suitable for each of the above administration forms can be taken. For example, in the case of an injection containing an anti-mucin antibody, the injection can be prepared by a conventional method, for example, after dissolving in an appropriate solvent (buffer solution such as PBS, physiological saline, sterilized water, etc.) If necessary, it can be prepared by sterilizing by filtration with a filter or the like and then filling in an aseptic container. If necessary, a conventional stabilizer is added to the injection, and examples of the stabilizer include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol and the like. Further, for example, a solubilizing agent, an antioxidant, a soothing agent, an isotonic agent and the like may be contained as necessary. The preparation form is preferably a preparation from which water has been removed by cryopreservation or freeze-drying, and the freeze-dried preparation is redispersed by adding purified water for injection at the time of use.

  An effective dose and administration schedule of the B cell injury inhibitor of the present invention can be appropriately determined by those skilled in the art from experience. The dose of the anti-mucin antibody can be appropriately adjusted depending on the degree of the disease, the age of the patient, the body weight, etc., but is usually selected from the range of 0.001 mg to 20 mg / kg / body weight per day, and the preferred range is 0. 0.01 mg to 10 mg / kg body weight, more preferably 0.05 to 1 mg / kg body weight, still more preferably 0.1 to 0.5 mg / kg body weight, once or several times a day It is administered in divided doses or continuously.

  The B-cell injury inhibitor of the present invention is used for the prevention and treatment of various diseases that occur or are thought to occur due to B-cell injury or reduced function. For example, it may be used for prevention / treatment of autoimmune diseases as well as prevention / treatment of decline in immune function of cancer-bearing patients.

  The B cell injury inhibitor of the present invention can also be used as an animal drug for mammals other than humans. Examples of the target mammal include livestock (for example, pigs, cows, horses, sheep, rabbits, etc.), companion animals (for example, dogs, cats, etc.), and examples of the diseases include the above-mentioned diseases. it can.

  Hereinafter, although this invention is demonstrated in detail based on a comparative example, an Example, and a formulation example, this invention is not limited to these Examples.

Comparative Example 1
(1) Reduction of splenic marginal zone B cells 2 × 10 ⁶ cells of mouse breast cancer cell line TA3-Ha (mucin producing strain) or TA3-St (mucin non-producing strain) were transplanted into the abdominal cavity of A / J mice, respectively. On day 8, the spleen was removed and white blood cells were prepared. Next, the cells were stained with fluorescently labeled anti-CD1d antibody and anti-CD21 antibody and analyzed with a flow cytometer. The results are shown in FIG. In the figure, the area surrounded by a square is the spleen marginal zone B cell, and it is known that CD1d and CD21 are highly expressed in the spleen marginal zone B cell.
As shown in FIG. 1, spleen marginal zone B cells were significantly decreased in TA3-Ha-bearing mice, whereas TA3-St-bearing mice changed compared to negative control (PBS) mice. There was no. That is, spleen marginal zone B cells were decreased in TA3-Ha (mucin producing strain) tumor-bearing mice.

(2) Antibody production against T cell-independent antigen As in (1) above, TA3-Ha or TA3-St was transplanted into the abdominal cavity of A / J mice, and T cells were not isolated on the 5th and 7th days. 10 μg of TNP-Ficoll, a dependent antigen, was intravenously injected from the tail vein. Blood was collected on the 8th day, and anti-TNP antibody in the serum was detected by the following method.
Serum diluted with PBS containing 5% BSA was added to a plate on which TNP-BSA had been immobilized, and incubated at room temperature for 1 hour. After washing, peroxidase-labeled anti-mouse IgM antibody or anti-mouse IgG antibody was added and further incubated at room temperature for 1 hour. After washing the plate again, color was developed by adding TMB substrate. After reacting for 30 minutes, sulfuric acid was added to stop the reaction, and the absorbance was measured. The result is shown in FIG.
As shown in FIG. 2, in the TA3-Ha tumor-bearing mice, the immune response to the T cell-independent antigen was decreased.

Example 1
Inhibition suppression of tumor-bearing mouse spleen marginal zone B cells by anti-mucin antibody treatment As in Comparative Example 1, TA3-Ha was transplanted into the abdominal cavity of A / J mice on days 3, 5 and 7 5 μg of MLS128 (anti-Tn antibody, anti-mucin monoclonal antibody prepared using human intestinal cancer cell LS180 as an immunogen) was administered from the tail vein. As a control, a mouse IgG antibody (Ctl Ab) of the same subclass was similarly administered.
On the eighth day, the spleen was removed to prepare white blood cells, and the cells were stained with fluorescently labeled anti-CD1d antibody and anti-CD21 antibody, and then analyzed with a flow cytometer. The result is shown in FIG.
As shown in FIG. 3, in the TA3-Ha tumor-bearing mice administered with MLS128, it was revealed that the decrease in splenic marginal zone B cells was suppressed, and the damage of B cells was suppressed. Considering the results of Comparative Example 1- (2), it was shown that the immune response was maintained even in TA3-Ha-bearing mice by administering the anti-mucin antibody.

Formulation Example 1
Aseptically prepare an aqueous solution containing 100 mg of MLS128 (anti-Tn antibody) and 100 mg of human serum albumin in 100 ml of 0.02 M phosphate buffer (containing 0.15 M NaCl and 0.01% polysorbate 80, pH 7.4). After dispensing, the product was freeze-dried and sealed to obtain a freeze-dried preparation.

It is a figure which shows the change of the spleen marginal zone B cell in Comparative Example 1- (1). In the figure, the area surrounded by a square is the spleen marginal zone B cell. It is a figure which shows the antibody production with respect to the T cell independent antigen in Comparative Example 1- (2). FIG. 3 shows changes in splenic marginal zone B cells in Example 1. In the figure, the area surrounded by a square is the spleen marginal zone B cell.

Claims (4)

  A B cell injury inhibitor containing an anti-mucin antibody as an active ingredient.   The B cell injury inhibitor according to claim 1, wherein the anti-mucin antibody is an antibody against epithelial mucin.   The B cell injury inhibitor according to claim 1 or 2, wherein the anti-mucin antibody is an antibody that recognizes a sugar chain portion of mucin.   The B cell injury inhibitor according to any one of claims 1 to 3, wherein the anti-mucin antibody is a monoclonal antibody.

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