JPS63152330A - Cellulicidal modified immunoglobulin - Google Patents

Abstract

PURPOSE:To obtain the titled and modified immunoglobulin having remarkably with activity, by linking an immunoglobulin or fragment thereof with a methotrexate through a specific disulfide linkage. CONSTITUTION:A cellulicidal modified immunoglobulin expressed by formula I (Ab is immunoglobulin or fragment thereof; CO linked with X is carbonyl originated in carboxyl or methotrexate; X is methotrexate residue; S is S which is introduced from itself or the outside of the immunoglobulin to the immunoglobulin; n is 1-30). The above-mentioned globulin is obtained, for example by reacting the immunoglobulin having an active disulfide group expressed by formula II (Y is organic group capable of forming active disulfide group together with a linked S; m is 1-30) with a cysteinamide derivative of methotrexate expressed by formula III.

Description

[Detailed description of the invention] <Industrial application field> The present invention relates to novel cell-killing modified immunoglobulins.

More specifically, the present invention relates to a novel cell-killing immunoglobulin in which methotrexate is bound via a disulfide bond to an immunoglobulin that selectively binds to a specific antigen of cells to be killed, or to a fragment thereof containing an antigen-binding site.

<Prior Art> As an example of obtaining a cell-killing 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 A chain of the toxin of the plant castor bean is used. , conjugates bound to immunoglobulin fragments.

(Refer to JP-A-55-136235 and JP-A-56-16418). In addition, as a related technology, methotrexate is
It has been reported that a cell-killing conjugate was obtained by converting the mercaptoethylamide derivative into a poly-D-lysine via a disphite bond [Shen et al.
et al., Journal of Biological Chemistry (J, Biol, Chem, ).

Volume 260, 10905-10908.1985]
. In these examples, the cell-killing effect is achieved because the disulfide bond linking 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 also discovered that even in the case of a methotrexate complex with an immunoglobulin or its fragment produced via a disulfide bond, there are differences in activity depending on the type of disulfide bond. When methotrexate was converted into a cysteine amide derivative and the thiol of the cystic acid group was used for the bond, methotrexate was converted into a 2-mercaptoethylamide derivative and the thiol group of the 2-mercaptoethylamine residue was used for the bond. We found that we could obtain a complex with significantly higher activity than in the case of conventional methods.

<Means for Solving the Problems> Accordingly, the present invention provides a compound of the general formula [Eco[Ab represents an immunoglobulin or a 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.

] It is a cell-killing modified immunoglobulin represented by

In the present invention, immunoglobulin refers to cells to be killed (
An immunoglobulin that can selectively bind to a specific antigen possessed by target cells (hereinafter referred to as target cells). Such immunoglobulin is produced, for example, as follows. i.e. monkeys, horses immunized with target cells such as tumor cells or specific lymphocytes.

Manufactured from antiserum isolated from animals such as cows, goats, sheep, rabbits, and chickens by known means such as ethanol fractionation, ammonium sulfate fractionation, ion exchange, molecular sieve column chromatography, protein A-Sepharose column chromatography, etc. be done. Alternatively, for example, lymphocytes collected from an animal immunized with target cells may be fused with transformed cells transformed with a virus, or lymphocytes with myeloma cells, etc. to obtain fused cells (hybridoma). 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 I (]
G, I (] A.

It is known that there are five types: I(]M, IoD, and IgE, any of which can be used in the present invention.

In the present invention, the immunoglobulin may be used as it is or as a fragment thereof [for example, Fab] as long as it contains a portion capable of binding to an antigen.

Fab', F (ab') 2 ] can also be used.

In general formula [I], 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 a methotrexate residue represented by the formula % or the formula. Moreover, -N HCHCH2S- COH in the general formula [I] is a residue derived from cysteine.

S bonded to Ab in general formula [I] may be a sulfur atom of the immunoglobulin or its fragment itself, or a sulfur atom introduced into the immunoglobulin or its fragment from the outside. That is,
The sulfur atom of a thiol group or Fab' of an immunoglobulin is produced by reductive cleavage of a disphite group of an immunoglobulin or a fragment thereof with a mercapto reagent such as 2-mercaptoethanol or dithiothreitol.
The sulfur atom of the immunoglobulin or its fragment itself, such as the hinge region of the fragment, or the sulfur atom of the thiol group of the J chain of the IgM immunoglobulin monomer [Ms, or, for example, N-succinimidyl 3-( It may also be a sulfur atom based on a disulfide group or thiol group of 2-pyridyldithio)propionate that is externally introduced into the immunoglobulin or its fragment using a disulfide group or thiol group-introducing agent.

The cytocidal cell-modified immunoglobulin represented by the general formula [I] of the present invention is, for example, the general formula []I], (Y-8+%A
b...[I[][Definitions of Ab and S are the same as in formula [I]. Y represents an organic group that forms an active disulfide group together with the sulfur atom bonded to Y. m represents an integer from 1 to 30. The immunoglobulin or fragment thereof having an active disphite group represented by the general formula [I], X -CO
N HCHCH2S H--...-[m]O2H [Definition of X and CO bonded to Formula % [ ] [Definition of CO bonded to × and X is the same as in formula [I]. The definition of Y is the same as in formula [II]. ] The cysteine amide active disulfide derivative of methotrexate has the general formula [V], (H3-) Ab
・・・・・・[V]勺L [Definition of Ab is the same as formula [I]. The definitions of S and m are the same as in formula [II]. ] 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 cell-killing modified immunoglobulins represented by formula [E], an immunoglobulin having an active disulfide group represented by formula [II] or a fragment thereof or methotrexate represented by formula [IV] is used. 1 to 100 moles of a cysteine amide derivative of methotrexate represented by formula [I], an immunoglobulin having a thiol group represented by formula [V], or a fragment thereof, per 1 mole of the cysteine amide active disulfide derivative of It is preferable to use 0.01 to 1 mol, and the reaction is preferably carried out using an immunoglobulin or a fragment thereof having an active disulfide group, or an immunoglobulin or a fragment thereof having a thiol group in 1) H5-9 buffer. solution(
The protein concentration is preferably adjusted to 0.1-40 mg/ml) at 0-37°C with stirring, respectively.
0 containing a small amount of solvent, e.g. 0.1M sodium chloride.
．． A cysteine amide derivative of methotrexate dissolved in IM phosphate buffer or N,N-dimethylformamide.

Cysteinamide activated disphite derivative of methotrexate is added and carried out for 15 minutes to 48 hours. Thereafter, by gel filtration or dialysis of the reaction mixture, unreacted cystinamide derivatives of methotrexate or cystinamide-activated disphite derivatives of methotrexate and low-molecular reaction products can be removed to purify the cell-killing modified immunoglobulin. .

The immunoglobulin or fragment thereof having a thiol group represented by the general formula [V] used in the above-mentioned method for producing a cell-killing modified immunoglobulin may be an immunoglobulin or a fragment thereof containing 2-iminothiolactone, N-acetylhomocysteine, etc. A thiol group is introduced using a thiol group-introducing agent such as thiolactone, or a disulfide group is introduced using a disulfite group-introducing agent such as N-succinimidyl 3-(2-pyridyldithio)propionate, and then a disulfide group is introduced into 2-mercaptoethanol. , can be obtained by converting it into a thiol group using a mercapto reagent such as dithiothreitol. In addition, the F ab' fragment of immunoglobulin, which originally has a thiol group, and the I(] MS fragment can be directly converted to the general formula [
It can be used as an immunoglobulin having a thiol group represented by V] or a fragment thereof.

The immunoglobulin or fragment thereof having an active disphite group represented by the general formula [II] is an immunoglobulin or a fragment thereof originally having a thiol group,
Alternatively, it can be obtained by reacting an immunoglobulin or a fragment thereof having a generated or introduced thiol group with a 2-biridyldisulfubis(2-nitrobenzoic acid)=11-disulfide compound.

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 cystine amide derivative of methotrexate represented by the formula [I[[] can be obtained by condensing methotrexate and cystine diester, followed by hydrolysis of the ester and reduction of the disulfide group with dithiothreitol.

The cysteine-activated disphite derivative of methotrexate represented by formula [IV] is a cysteine derivative of methotrexate that is converted into 2-pyridyl disulfide, 5.5'
It can be obtained by reacting with an active disulfide compound such as -dithiobis(2-nitrobenzoic acid).

<Examples and reference examples> The present invention will be explained in detail below using reference examples and examples.

Reference example Synthesis of cysteine amide derivative of methotrexate
L-cystine dimethyl ester 2H was added to the resulting solution under water cooling.
Cu powder 270R9 was added to 0. IN N a OH15,
8#11! A solution obtained by dissolving the solution was added, and a 2N HC aqueous solution was added dropwise to the resulting solution to adjust the pH of the solution to 6.70.

Add 190% of 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride to this solution while cooling with water and stirring.
Rng powder was added, the water bath was removed, and the mixture was stirred at room temperature for 15 hours. The resulting precipitate was filtered and added to 0.1M sodium phosphate buffer (1)H7,4) 50m! ! and ethanol 1
After washing it at 0m, drying it under reduced pressure to make powder 328ryJ.
I got it. The powder was added to 17.3 d of 0.1 N NaOH to form a suspension and stirred at room temperature for 5 hours. As the reaction proceeded, it gradually dissolved and became a uniform yellow solution. Add 0.2N SC, Q to bring the l)H of the solution to 8.5
2 ml of a 1012M sodium borate buffer solution was added, and then 265.7 Rg of dithiothreitol powder was added and heated at 50° C. for 30 minutes. Next, cool the solution on ice and
NHCu and 0. Add IN HCu to bring the pH of the solution to 3.
．． 95 and left to stand for 1 hour, the resulting precipitate was collected by filtration.

The precipitate was washed with 507 liters of water and 20 Id of ethanol and dried under reduced pressure to obtain 200 rRg of a cysteine amide derivative powder of mesomerexate (yield 54.4%).

Physicochemical properties m, p,: 195-201°C (color change decomposition) Twill desorption mass spectrometry (FD-MS): M+H+
558, nibr IR, U 3350 (S, br), 2550 (s
h).

1640 (S), 1540 (S) cm”
Example 1 (a) Production of cell-killing modified immunoglobulin Mouse breast cancer M
Mouse monoclonal antibody 1 against M46 cells (
2810 mg of lG was added to 0.0 mg containing 0.1 M sodium chloride.
．． 1M phosphate buffer (pH 7.5) - (hereinafter referred to as phosphate buffer) After dissolving in 1.4mM, an ethanol solution containing 16mM N-succinimidyl 3-(2-pyridyldithio)probionate was added to 0.4M phosphate buffer. 05'd was gradually added, stirred, and reacted at 25°C for 30 minutes. The reaction solution was thoroughly dialyzed against the above phosphate buffer to obtain IgG2a having an average of 5.6 3-(2-pyridyldithio)propionyl groups per molecule.

Then, the thus obtained 3-(2-pyridyldithio)
Phosphate buffer 1 containing propionylated I(]G2aRg
．． 2#ll! 0.05 d of phosphate buffer containing 16 mg of cysteine amide derivative o of methotrexate was added to the solution, stirred, and allowed to react at room temperature for 17 hours. It was thoroughly dialyzed against buffer solution (IIH7,2).

In this way, 5.9 ml of IgG2a (cell-killing modified immunoglobulin) having an average of 4.2 cysteine amide derivatives of methotrexate bound per molecule was obtained.

(0) Culture of cell-killing modified immunoglobulin against tumor cells Cell-killing mouse breast cancer MM46 cells at 2.5X 104 cells/at
! RPM I containing 10% fetal bovine serum so that
Suspend in 1640 medium and add 0 to each well of a 96-well flat bottom plate.
．． 2Id each was dispensed. Next, 0.02 m of dissolved cell-killing modified immunoglobulin (produced in (a) above) at various concentrations was added to the above medium, stirred, and incubated at 37°C in a 5% CO2 atmosphere. Cultured for 1 day. The above operations were performed aseptically.

After culturing, 0.02 m 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 cell-killing modified immunoglobulin (A in Table 1) produced in (a) showed strong cell-killing activity at concentrations exceeding 0.01 μM equivalent to methotrexate. In contrast, a modified immunoglobulin conjugated with a 2-mer putethylamide derivative of methotrexate (
B) in Table 1 showed only weak cell killing activity.

That is, it was found that this modified immunoglobulin had about 30 times stronger cell killing activity.

Table 1 A: Modified immunoglobulin conjugated with cysteine amide derivative of methotrexate B: Modified immunoglobulin conjugated with 2-mercaptoethylamide derivative of methotrexate (comparative example) Example 2 Anti-MM46 modified immunoglobulin, non-specific modified immunoglobulin Comparison of cell killing properties of globulins and cysteine amide derivatives of methotrexate A cysteine amide derivative of methotrexate was prepared in the same manner as in Example 1 (a) using normal (non-specific) mouse immunoglobulin in place of the anti-MM46 monoclonal antibody. A non-specifically modified immunoglobulin having an average of 3.4 molecules bound per immunoglobulin molecule was obtained.

Example 1 Regarding the anti-MM46 cell-killing modified immunoglobulin, non-specifically modified immunoglobulin, and cysteine amide derivative of methotrexate produced in Example 1 (a)
The effect on cultured MM46 cells was examined as in (O).

The results are summarized in Table 2. The cytocidal modified immunoglobulin exhibited cell killing at much lower concentrations than the other two.

Table 2

Claims (1)

[Claims] General formula [I] ▲ Numerical formulas, chemical formulas, tables, etc. are included ▼ [I] [Ab represents immunoglobulin or a 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 to which Ab is attached 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. ] A cell-killing modified immunoglobulin represented by: