JPH053856B2 - - Google Patents

Description

[Detailed description of the invention]

<Industrial Application Field> The present invention relates to an antitumor agent containing a novel modified immunoglobulin as an active ingredient. More specifically, the active ingredient is a novel immunoglobulin in which methotrexate is bound via a disulfide bond to an immunoglobulin that selectively binds to a specific antigen of tumor cells or a fragment thereof containing an antigen-binding site. Regarding antitumor agents. <Prior Art> As an example of obtaining an antitumor immunoglobulin by bonding a biologically active substance to an immunoglobulin or a fragment thereof via a disulfide bond, for example, fragment A of diphtheria toxin or chain A of the toxin of the plant castor bean is , conjugates bound to immunoglobulin fragments. (Refer to Japanese Patent Application Laid-Open Nos. 55-136235 and 56-16418). In addition, as a related technology, methotrexate is
It has been reported that an antitumor conjugate was obtained by converting the mercaptoethylamide derivative into poly-D-lysine via a disulfide bond [Shen et al., Journal of Biological Chemistry]. J.Biol.Chem.), 260
Vol. 10905-10908, 1985]. In these examples,
The antitumor effect is achieved because the disulfide bond that connects the carrier polymer and the biologically active substance is efficiently cleaved within the cell to release the biologically active substance. <Problems to be Solved by the Invention> The present inventors have discovered that even in the case of a methotrexate complex with an immunoglobulin or its fragment produced via a disulfide bond, the activity differs depending on the type of disulfide bond. We found that if methotrexate was converted into a cysteinamide derivative and then the thiol of the cysteic acid group was used for the bond, then methotrexate was converted into a 2-mercaptoethylamide derivative and the thiol group of the 2-mercaptoethylamine residue was used for the bond. It was found that a complex with significantly higher activity could be obtained than when using this method. <Means for solving the problems> Therefore, the present invention provides the general formula [] [Ab stands for immunoglobulin or fragment thereof. The CO bonded to X is a carbonyl group derived from the carboxyl group of methotrexate. X represents a methotrexate residue.
The S attached to the Ab is a sulfur atom of the immunoglobulin or fragment thereof itself or a sulfur atom introduced into the immunoglobulin or fragment thereof from outside. n represents an integer from 1 to 30. ] This is an antitumor agent containing a modified immunoglobulin represented by the following as an active ingredient. In the present invention, immunoglobulin refers to immunoglobulin that can selectively bind to a specific antigen possessed by tumor cells (hereinafter referred to as target cells). Such immunoglobulin is produced, for example, as follows. That is, ethanol fraction, ammonium sulfate fraction, and ion fraction are obtained from antiserum isolated from animals such as monkeys, horses, cows, goats, sheep, rabbits, and chickens that have been immunized with target cells such as tumor cells or specific lymphocytes. It is produced by known means such as exchange, molecular sieve column chromatography, and protein A-Sepharose column chromatography. Alternatively, for example, lymphocytes collected from an animal immunized with target cells can be transformed with a virus to transform cells, or lymphocytes can be fused with myeloma cells to obtain fused cells (hybridoma), which can be used to produce antibodies. After selecting productive clones, the immunoglobulin used in the present invention can be obtained from the culture medium obtained by culturing the cells, or from the serum or peritoneal fluid obtained by inoculating these clones into animals. In addition to the target cells, antigens used for immunization to obtain the above-mentioned antiserum or antibody-producing lymphocytes include antigenic substances extracted from the target cells, or artificially synthesized antigens of the target cells. You can also use Furthermore, in the production of the above immunoglobulin, antigen-sensitized lymphocytes obtained from humans and treated with antigenic substances as necessary may be used as antibody-producing lymphocytes used for transformation or cell fusion. I can do it. Immunoglobulins include IgG, IgA, IgM,
It is known that there are five types of IgD and IgE.
Any of them can be used in the present invention. In the present invention, the immunoglobulin can be used as it is or as a fragment thereof [e.g., Fab,
Fab′, F(ab′) ₂ ] can also be used. In the general formula [ ], CO bonded to X
is a carbonyl group derived from the carboxyl group of methotrexate, and X is a methotrexate residue. That is, X is the formula This is a methotrexate residue represented by Also, in the general formula [] is a residue derived from cysteine. In the general formula [], S bonded to Ab is
It may be a sulfur atom of the immunoglobulin or fragment thereof itself or a sulfur atom introduced into the immunoglobulin or fragment thereof from an external source. That is, the disphite group of an immunoglobulin or a fragment thereof is, for example, 2
- The sulfur atom of a thiol group produced by reductive cleavage with a mercapto reagent such as mercaptoethanol or dithiothreitol, or the sulfur atom of an immunoglobulin.
The sulfur atom of the immunoglobulin or its fragment itself, such as the hinge region of a Fab' fragment, or the sulfur atom of the thiol group of the J chain of the monomeric IgMs of an IgM immunoglobulin, or, for example, N-succinimidyl 3- (2-pyridyldithio)propionate 2-iminothiolactone

[Formula] N- Acetylhomocysteine thiolactone

It may be a sulfur atom based on a disulfide group or a thiol group that is externally introduced into an immunoglobulin or a fragment thereof using a disulfide group or thiol group-introducing agent such as the following formula. The antitumor cell-modified immunoglobulin of the present invention represented by the general formula [] is, for example, the general formula [], (Y-S) _-n Ab...[] [Definitions of Ab and S are the same as the formula [] . Y represents an organic group which forms an active disulfide group together with the sulfur atom bonded to Y. m is 1~30
represents an integer. ] The immunoglobulin or fragment thereof having an active disphide group represented by the general formula [], [The definition of X and CO bonded to X is the formula []
Same as . ] or by reacting a cysteinamide derivative of methotrexate represented by the general formula [], [Definitions of X and CO bonded to X are the same as in formula []. The definition of Y is the same as in formula []. ] The cysteine amide active disulfide derivative of methotrexate has the general formula [], (HS) _-n Ab...[] [The definition of Ab is the same as the formula []. The definitions of S and m are the same as in formula []. ] It can be produced by reacting an immunoglobulin having a thiol group represented by the following or a fragment thereof. In these methods for producing antitumor modified immunoglobulin represented by the formula [], the formula []
For each mole of the immunoglobulin or its fragment having an active disulfide group represented by the formula [] or the cysteinamide active disulfide derivative of methotrexate represented by the formula [], the cysteine amide derivative of methotrexate represented by the formula [], the formula 1 to 100 mol or 0.01 to 100 mol of an immunoglobulin or a fragment thereof having a thiol group represented by [ ]
It is preferable to use 1 mol, and the reaction is preferably carried out using a solution of an immunoglobulin or a fragment thereof having an active disulfide group, or an immunoglobulin or a fragment thereof having a thiol group, in a buffer having a pH of 5 to 9 (the protein concentration is preferably (adjusted to 0.1-40 mg/ml) and a small amount of solvent, e.g., while stirring at 0-37°C.
A cysteinamide derivative of methotrexate or a cysteinamide active disulfide derivative of methotrexate dissolved in a 0.1M phosphate buffer containing 0.1M sodium chloride or N,N-dimethylformamide is added, and the reaction is carried out for 15 minutes to 48 hours. Thereafter, the reaction mixture is subjected to gel filtration or dialysis to remove unreacted cysteine amide derivatives of methotrexate or cysteine amide activated disulfide derivatives of methotrexate and low-molecular reaction products, thereby purifying the antitumor modified immunoglobulin. I can do it. The immunoglobulin or fragment thereof having a thiol group represented by the general formula [] used in the above method for producing an anti-tumor modified immunoglobulin is an immunoglobulin or a fragment thereof which is injected with 2-iminothiolactone, N-acetylhomocysteine thio A thiol group is introduced using a thiol group-introducing agent such as lactone, or a disulfide group is introduced using a disulfide group-introducing agent such as N-succinimidyl 3-(2-pyridyldithio)propionate, and 2-mercaptoethanol, It can be obtained by converting it into a thiol group using a mercapto reagent such as dithiothreitol. Furthermore, Fab' fragments and IgMs fragments of immunoglobulins that originally have a thiol group can be used as they are as immunoglobulins that have a thiol group represented by the general formula [ ] or fragments thereof. An immunoglobulin or a fragment thereof having an active disulfide group represented by the general formula [] is an immunoglobulin or a fragment thereof having an originally thiol group, or an immunoglobulin or a fragment thereof having a thiol group generated or introduced. -pyridyl disulfide

[Formula] 4-pyridyl disulfide

[Formula] 5,5'-dithiobis(2-nitrobenzoic acid)

It can be obtained by reacting an active disulfide compound such as [Formula]. It can also be obtained by directly reacting an active disulfide group-introducing agent such as N-succinimidyl 3-(2-pyridyldithio)propionate with immunoglobulin or a fragment thereof. The cysteinamide derivative of methotrexate represented by the formula [] can be obtained by condensing a diester of methotrexate and cystine, followed by hydrolysis of the ester and reduction of the disulfide group with dithiothreitol. The cysteine-activated disulfide derivative of methotrexate represented by the formula [] is a cysteine derivative of methotrexate that is converted into 2-pyridyl disulfide, 5,5'-dithiobis(2-nitrobenzoic acid)
It can be obtained by reacting with an active disulfide compound such as. <Examples and Reference Examples> The present invention will be explained in detail below using reference examples and examples. Reference Example Synthesis of Cysteinamide Derivative of Methotrexate 300 mg of methotrexate powder was mixed into approximately 12 ml of
Add L-cystine dimethyl ester to the resulting solution under ice cooling.
A solution obtained by dissolving 270 mg of 2HCl powder in 15.8 ml of 0.1N NaOH was added, and an aqueous 2NHCl solution was added dropwise to the resulting solution to adjust the pH of the solution to 6.70. Add 1-ethyl-3- to this solution under ice-cooling and stirring.
A powder of 190 mg of (3-dimethylamino-propyl)carbodiimide hydrochloride was added, and the ice bath was removed and the mixture was stirred at room temperature for 15 hours. The resulting precipitate was filtered and 0.1M
Wash this with 50ml of sodium phosphate buffer (PH7.4) and 10ml of ethanol, then dry under reduced pressure to form a powder.
Obtained 328 mg. The powder was added to 17.3 ml of 0.1N NaOH to obtain suspension and stirred at room temperature for 5 hours. As the reaction progressed, it gradually dissolved and became a uniform yellow solution. Add 0.2NHCl to adjust the pH of the solution
After adjusting to 8.5, add 2 ml of 0.2M sodium borate buffer.
and then 265.7 mg of dithiothreitol powder.
was added and heated at 50°C for 30 minutes. Next, cool the solution on ice and add 2NHCl and 0.1NHCl to adjust the pH of the solution.
3.95 and left to stand for 1 hour, the resulting precipitate was collected by filtration. The precipitate was washed with 50 ml of water and 20 ml of ethanol and dried under reduced pressure to obtain 200 mg of a powder of cysteinamide derivative of mesomerexate (yield 54.4%). Physicochemical properties MP: 195-201℃ (discoloration and decomposition) Field desorption mass spectrometry (FD-
MS): M+H ⁺ 558 IR: U ^HB 〓 _nax 3350 (S, br), 2550 (sh), 1640 (S
),
1540 (S) cm ^-1 Example 1 (a) Production of antitumor modified immunoglobulin 10 mg of mouse monoclonal antibody IgG2a against mouse breast cancer MM46 cells was added to 0.1 M phosphate buffer (PH7.5) containing 0.1 M sodium chloride ( After dissolving in 1.4 ml of phosphate buffer (hereinafter referred to as phosphate buffer), 16 mM N-succinimidyl 3-(2-pyridylthio)
0.05 ml of an ethanol solution containing propionate was gradually added, stirred, and reacted at 25° C. for 30 minutes. The reaction solution was thoroughly dialyzed against the above phosphate buffer, and 3
IgG2a having an average of 5.6 -(2-pyridyldithio)propionyl groups per molecule was obtained. Next, 0.05 ml of a phosphate buffer containing 0.16 mg of the cysteinamide derivative of methotrexate was added to 1.2 ml of the phosphate buffer containing the 3-(2-pyridyldithio)propionylated IgG2amg obtained in this way, and the mixture was stirred and brought to room temperature. After reacting for 17 hours, the reaction solution was thoroughly dialyzed against 10 mM phosphate buffer (PH7.2) containing 0.14 M sodium chloride. In this way, IgG2a bound to an average of 4.2 cysteinamide derivatives of methotrexate per molecule
(Antineoplastic modified immunoglobulin) 5.9 mg was obtained. (b) Cell killing of cultured tumor cells with anti-tumor modified immunoglobulin Mouse breast cancer MM46 cells were suspended in RPMI1640 medium containing 10% fetal bovine serum to a concentration of 2.5 x 10 ⁴ cells/ml and placed in a 96-well flat bottom plate. 0.2 ml was dispensed into each hole. Next, 0.02 ml of dissolved anti-tumor modified immunoglobulin (manufactured in (a) above) at various concentrations was added to the above medium, stirred, and kept at 37°C for 3 days in a 5% CO ₂ atmosphere. Cultured. The above operations were performed aseptically. After culturing, 0.02 ml of 3% trypan blue solution was added, and living cells that were not stained with trypan blue were counted under a microscope. The results are summarized in Table 1. The antitumor modified immunoglobulin produced in (A) (A in Table 1), at a concentration exceeding 0.01 μM equivalent to methotrexate,
It showed strong cell killing activity. In contrast, the modified immunoglobulin to which the 2-mercaptoethylamide derivative of methotrexate was bound (B in Table 1) showed only a weak cell-killing activity. That is, it was found that this modified immunoglobulin had about 30 times stronger cell killing activity.

[Table] Modified immunoglobulin with conductor attached
B: Modified immunoglobulin conjugated with 2-mercaptoethylamide derivative of methotrexate (comparative example)
Example 2 Comparison of cell killing properties of anti-MM46 modified immunoglobulin, non-specific modified immunoglobulin, and cysteinamide derivative of methotrexate Example 1 was performed using normal (non-specific) mouse immunoglobulin instead of anti-MM46 monoclonal antibody.
A non-specifically modified immunoglobulin having an average of 3.4 cysteinamide derivatives of methotrexate bound per immunoglobulin molecule was obtained by the same method as in (a). The anti-MM46 antitumor modified immunoglobulin, non-specifically modified immunoglobulin, and methotrexate cysteinamide derivative produced in Example 1 (a) were tested for their effects on cultured MM46 cells as in Example 1 (b). Examined. The results are summarized in Table 2. The anti-tumor modified immunoglobulin exhibited cell killing at much lower concentrations than the other two.

【table】

Claims (1)

[Claims] 1. General formula [] [Ab stands for immunoglobulin or fragment thereof. The CO bonded to X is a carbonyl group derived from the carboxyl group of methotrexate. X represents a methotrexate residue.
The S attached to the Ab is a sulfur atom of the immunoglobulin or fragment thereof itself or a sulfur atom introduced into the immunoglobulin or fragment thereof from the outside. n represents an integer from 1 to 30. ] An antitumor agent containing a modified immunoglobulin represented by the following as an active ingredient.