Antibody specifically recognizing immunosuppressant and method for producing the same

Abstract

To provide a method for producing an antibody specifically recognizing an immunosuppressant because an immunosuppressant is generally a low molecular weight compound, and it is extremely difficult to acquire an antibody even if it is immunized alone to an animal.SOLUTION: A composition containing an immune complex created by bonding an immunosuppressant, a linker and a carrier protein in this order, and a metabolic inhibitor of immunosuppressant is immunized to an animal except for human, and then a produced antibody is collected from the animal to obtain an antibody specifically recognizing immunosuppressant.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an antibody that specifically recognizes an immunosuppressant and a method for producing the same. [Background technology]

[0002] Immunosuppressants are used to suppress rejection after organ transplants and to treat autoimmune diseases, allergic diseases, or rheumatic diseases. Non-Patent Document 1 reports that immunosuppressants have a narrow therapeutic concentration range and are known to have serious side effects such as kidney damage. In order to administer an appropriate dose, it is necessary to measure the blood concentration of immunosuppressants. Patent Document 1 discloses a method for quantifying immunosuppressants contained in blood, etc., using ELISA (Enzyme Linked Immunosorbent Assay). To perform enzyme immunoassay, an antibody that specifically recognizes the immunosuppressant is required. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 3551431 [Non-patent literature]

[0004] [Non-Patent Document 1] Okayama Medical Association Journal, Vol. 119, p. 199-203, September 3, 2007 (Published: July 4, 2008) Summary of the Invention [Problem to be solved by the invention]

[0005] Immunosuppressants are generally low-molecular-weight compounds, and therefore antibodies cannot be obtained by immunizing animals alone. Therefore, a common method for obtaining antibodies is to immunize experimental animals such as mice or rats with a complex (hereinafter referred to as an "immune complex") composed of an immunosuppressant, a linker, and a carrier protein bound in this order. Meanwhile, there are various cross-reactive substances for antibodies that recognize immunosuppressants. For example, cyclosporine (hereinafter referred to as "Cs"), an immunosuppressant represented by formula (I), is metabolized to metabolites such as AM1 represented by formula (II), AM4N represented by formula (III), and AM9 represented by formula (IV), which are known as cross-reactive substances.

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[0010] The present inventors prepared an immunogen by introducing a linker into the 2-position of cyclosporine and performed immunization, but the resulting antibody strongly recognized the cross-reactive substance AM1. Generally, methods for preparing immunogens to obtain highly specific antibodies involve examining the position at which the linker is introduced. However, because the immunosuppressants cyclosporine and tacrolimus have unique structures, it is extremely difficult to introduce a linker into any desired position. [Means for solving the problem]

[0011] The present inventors have intensively investigated ways to solve the above problems, and as a result, have found that adding an immunosuppressant metabolic inhibitor to an immunizing antigen at the time of immunization inhibits the metabolism of the immunosuppressant in the body of the immunized animal, thereby maintaining the presence of the immunosuppressant, and thereby enabling the production of antibodies that specifically recognize the immunosuppressant, thereby completing the present invention.

[0012] That is, the present invention is as follows: (1) An immune complex comprising an immunosuppressant, a linker, and a carrier protein bound in this order; and Metabolic inhibitors of immunosuppressants A composition comprising: (2) An immunization method, comprising immunizing a non-human animal with the composition according to (1) above. (3) A method for producing an antibody that specifically recognizes an immunosuppressant, which comprises immunizing an animal by the method described in (2) above, and then recovering the produced antibody from the animal. (4) An antibody that specifically recognizes an immunosuppressant, produced by the method described in (3) above.

[0013] The present invention will be described in detail below.

[0014] In the present invention, the animal to be immunized is not particularly limited as long as it is an animal other than a human, and examples thereof include mice, rats, rabbits, chickens, and goats that are commonly used by those skilled in the art.

[0015] Immunosuppressants that can be used in the present invention have at least one site that can be bound to a linker. Preferably, the immunosuppressant produces a cross-reactive substance through metabolism. Examples include steroids, azathioprine, methotrexate, mizoribine, mitoxantrone, cyclosporine, and tacrolimus. Of these, cyclosporine and everolimus are preferred. This is because their metabolites are not involved in the mechanism of action, and it is therefore important to produce an antibody that specifically recognizes the immunosuppressant but not the metabolite, and measure the blood concentration of the immunosuppressant.

[0016] The immunosuppressant metabolic inhibitor used in the present invention is not particularly limited as long as it is a drug that inhibits the metabolism of immunosuppressants, and examples thereof include azole antifungal drugs (ketoconazole, itraconazole, fluconazole) that inhibit the metabolic enzyme CYP3A4, macrolide antibiotics (erythromycin, clarithromycin, triacetyloleandomycin), verapamil, cimetidine, and ethinylestradiol.

[0017] The linker used in the method of the present invention, which mediates the bond between the immunosuppressant and the carrier protein, is not particularly limited as long as it does not completely impair the interaction between the resulting antibody and the labeled immunosuppressant. NHS (N-hydroxysuccinimide) compounds, which are reactive with amino groups, are often used due to their ease of availability and low cost. Other linkers that can be used include compounds reactive with amino groups, such as imidoester compounds; compounds reactive with carboxyl groups, such as carbodiimide compounds; and compounds reactive with thiol groups, such as maleimide compounds, haloacetyl compounds, and pyridylacetyl compounds. Furthermore, non-covalent bonds with high affinity, such as avidin-biotin bonds, may also be used as linkers.

[0018] In the present invention, the carrier protein is not particularly limited, but is preferably an immunogenic polymer, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), blue carrier protein (BCP), or ovalbumin (OVA).

[0019] The mixing ratio of the immune complex to the metabolic inhibitor is not particularly limited, but is preferably 1:1 to 10:1 (weight ratio), more preferably 3:1 to 7:1 (weight ratio).

[0020] Immunization may be performed by any method commonly used in animal immunization, such as intraperitoneal administration, subcutaneous injection, or intramuscular injection, and is not particularly limited.

[0021] The amount of immune complex can be adjusted depending on the size of the animal to be immunized. Specifically, the amount of immune complex used per immunization is 50 to 100 micrograms for mice and 0.5 to 1 milligram for rabbits.

[0022] The number of immunizations varies depending on the type of immune complex and the responsiveness of the immunized animal, so it is preferable to determine the number of immunizations by analyzing the antibody titer by ELISA or the like.

[0023] After immunization in this manner, antibodies produced by the immunized animals can be collected to obtain antibodies capable of specifically recognizing the immunosuppressant. The collection method is not particularly limited, and any conventional collection method such as salting out or chromatography may be used as appropriate. [Effects of the Invention]

[0024] According to the present invention, an antibody capable of specifically recognizing an immunosuppressant can be obtained, and the antibody can be used to accurately measure the blood concentration of an immunosuppressant in a patient administered the immunosuppressant.

[0025] Cimetidine, a metabolic inhibitor of immunosuppressants, generally inhibits the intracellular metabolism of the immunosuppressant Cs. However, according to the present invention, it has been found that cimetidine inhibits the metabolism of Cs not only intracellularly but also extracellularly. This means that cimetidine inhibits the metabolism of Cs even when immunized extracellularly with CS as the immunogen. As a result, the present invention has the excellent effect of enabling the production of antibodies that specifically recognize the immunosuppressant Cs. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 shows B / B0 of the Cs-BSA / Cim immunized ICR antiserum of Example 2. [Figure 2] FIG. 1 shows the B / B0 ratio of the Cs-BSA immunized ICR antiserum of Comparative Example 1. [Figure 3]FIG. 1 shows the cross-reactivity rate evaluation of antisera obtained in Example 4. [Example]

[0027] The method for obtaining an antibody that specifically recognizes the immunosuppressant of the present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these.

[0028] Example 1 Preparation of Reagents (1) Preparation of immune complexes A compound represented by formula (V) in which bovine serum albumin and N-hydroxysuccinimide are introduced at the 2-position of cyclosporin via a linker.

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[0030] The two were mixed at a molar ratio of 1:5. Cyclosporine-labeled BSA (hereafter referred to as Cs-BSA) was recovered from the reaction mixture and used as an immune complex.

[0031] (2) Preparation of metabolic inhibitors Cimetidine (hereinafter referred to as Cim) was dissolved in ethanol to a concentration of 1 mg / ml.

[0032] (3) Preparation of labeled antigen Alkaline phosphatase (hereinafter referred to as ALP) and a compound represented by formula (V), in which N-hydroxysuccinimide is introduced at the 2-position of cyclosporine via a linker, were mixed at a molar ratio of 1:5. ALP labeled with cyclosporine (hereinafter referred to as Cs-ALP) was recovered from the reaction mixture and used in the following examples.

[0033] [Example 2] Immunization of experimental animals (Cs-BSA / Cim) First, mice (ICR, female, 4 weeks old) were prepared for immunization. Next, a 6:1:4.25 weight ratio mixture of 1.25 mg / ml immune complex (Cs-BSA), 1 mg / ml Cim, and Freund's complete adjuvant was prepared. This mixture was then emulsified by sonication on ice. 100 μl of this solution (equivalent to 50 μg immune complex and 8.3 μg Cim) was then injected subcutaneously into the prepared mice for immunization.

[0034] One week after the first immunization, mice were immunized again as follows. First, a 6:1:4.25 weight ratio mixture of 1.25 mg / ml immune complex (Cs-BSA), 1 mg / ml Cim, and Freund's incomplete adjuvant was prepared. This mixture was then emulsified by sonication on ice. 100 μl of this solution (equivalent to 50 μg immune complex and 8.3 μg Cim) was injected subcutaneously into the mice for immunization. Subsequent immunizations using Freund's incomplete adjuvant were repeated every other week for three months.

[0035] [Comparative Example 1] Immunization of experimental animals (Cs-BSA) First, mice (ICR, female, 4 weeks old) were prepared as immunized animals. Next, a 20:17 mixture (by weight) of 1 mg / ml immune complex (Cs-BSA) solution and Freund's complete adjuvant was prepared. This mixture was then emulsified by sonication on ice. 100 μl of this solution (equivalent to 50 μg of immune complex) was then injected subcutaneously into the prepared mice for immunization.

[0036] One week after the first immunization, a second immunization was performed as follows. First, a 20:17 mixture (by weight) of 1 mg / ml immune complex (Cs-BSA) solution and Freund's incomplete adjuvant was prepared. This mixture was then emulsified by sonication on ice. 100 μl of this solution (equivalent to 50 μg of immune complex) was then injected subcutaneously into a mouse for immunization. For the second and subsequent immunizations, this immunization procedure using Freund's incomplete adjuvant was repeated every other week for three months.

[0037] [Example 3] Antibody titer evaluation of antisera After 3 months of immunization, a small amount of blood was collected from each of the mice immunized in Example 2 and Comparative Example 1. Antisera were prepared from the blood and the cross-reactivity of the antisera with Cs and AM1 was evaluated by the ELISA described below. (1) Commercially available anti-mouse IgG (hereinafter referred to as αMs-IgG) (0.5 μg / mL) was immobilized on an ELISA plate, which was then blocked with 1% skim milk / PBS. (2) The antiserum was diluted 1000-fold with 0.1% skim milk and reacted with αMs-IgG on an ELISA plate. (3) After B / F separation, Cs-ALP (32 ng / mL) was reacted in the presence of cyclosporine at seven concentrations (15.6 ng / mL, 31.3 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL) or AM1 at seven concentrations (15.6 ng / mL, 31.3 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL). (4) After B / F separation, the enzyme substrate, 4-methylumbelliferyl phosphate (4-MUP), was added and detected by measuring the fluorescence intensity.

[0038] [Example 4] Evaluation of cross-reaction rate The signal (B) in the presence of cyclosporine or AM1 at the seven concentrations was calculated as a relative value (B / B [%]) when Cs-ALP was added in the absence of cyclosporine or AM1, with the signal (B) being 100%. The cyclosporine or AM1 concentration was plotted on the horizontal axis and B / B (%) on the vertical axis (Figure 1: Example 2, Figure 2: Comparative Example 1). In each of Figures 1 and 2, the cross-reactivity rate was calculated using the B / B value of 125 ng / mL, which was the median value of the seven concentrations. Cross-reaction rate (%) = [(B / B0 of Cs) / (B / B0 of AM1)] × 100 The results are shown in Figure 3. The antiserum obtained by immunization with Cs-BSA / Cim in Example 2 was found to have extremely low cross-reactivity with AM1 compared to the antiserum obtained by immunization with Cs-BSA in Comparative Example 1. These results demonstrate that immunization with Cs-BSA supplemented with cimetidine can yield anti-cyclosporine antibodies with extremely low cross-reactivity with AM1.

Claims

1. - an immune complex comprising an immunosuppressant, a linker, and a carrier protein bound in this order; and - Metabolic inhibitors of immunosuppressants A composition comprising:

2. An immunization method, comprising immunizing a non-human animal with the composition of claim 1.

3. A method for producing an antibody that specifically recognizes an immunosuppressant, comprising immunizing an animal by the method of claim 2 and then recovering the produced antibody from the animal.

4. An antibody that specifically recognizes an immunosuppressant, produced by the method of claim 3.

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