JPH0662438B2 - Cytotoxic antibody conjugate and method for producing same - Google Patents

Description

[Detailed Description of the Invention] Technical Field The present invention relates to cytocidal antibody conjugates.

More specifically, the present invention relates to a cytotoxic antibody conjugate in which an antifolate folate analog is bound via an oligopeptide to an antibody or a fragment thereof that selectively binds to a specific antigen on the cells to be killed, and to a method for producing the same.

BACKGROUND ART Folate antagonistic folic acid analogues are compounds that have a chemical structure similar to folic acid,
These substances partially or completely block the action of folic acid or its derivatives. Many such substances have been synthesized,
Some of them are used in chemotherapy for malignant tumors such as leukemia and cancer.

Regarding the cytotoxic antibody conjugates in which an antifolate folate analog is bound to an antibody, methods of binding methotrexate to the antibody using a water-soluble carbodiimide condensing agent and methods of binding using an active ester of methotrexate are known (P. N. Kulkarni et al., Cancer Research, 2004).
Cancer Res. 41, 2700-2706, 198
1 year). Also, a method is known in which methotrexate is bound to an antibody and serum albumin is used as an intermediate drug support (M. C. Garnett et al.,
International Journal of Cancer (Int. J. Cancer), Vol. 31, pp. 661-670, 19
1983). However, antibodies or their fragments,
There are no known methods for conjugating antifolate analogs via oligopeptides, nor are there any known cytotoxic antibody conjugates. Conjugates obtained by directly conjugating methotrexate to conventional antibodies lack sufficient potency and selectivity in cytotoxic and antitumor activity, and oligopeptide-mediated conjugates are expected to improve these points.

DISCLOSURE OF THE INVENTION The present inventors have conducted extensive research to develop a method for binding an antifolate folate analog to an antibody or its fragment via an oligopeptide, and to develop a useful cytotoxic antibody conjugate by this method. In the intermediate obtained by binding an oligopeptide to an antifolate folate analog at its N-terminus, there is a carboxyl group on the antifolate folate analog in addition to the C-terminus of the oligopeptide, and therefore it is not possible to selectively bind the oligopeptide to an antibody or its fragment via the former carboxyl group. Therefore,
It is necessary to develop a mode of reaction with the antibody or fragment thereof, including directing the intermediate to a suitable reactive derivative.

The present inventors have discovered that halogenated (especially iodinated and brominated) acetylhydrazide compounds are highly reactive with the amino groups of antibodies or their fragments, and that antifolate-active folate analogs can be derived from halogenated acetylhydrazide derivatives via oligopeptide linkages, thereby arriving at the present invention.

That is, the present invention relates to a cytotoxic antibody conjugate in which an antifolate folate analog is bound to an antibody or a fragment thereof via an oligopeptide, and preferably, the conjugate is represented by the general formula [I]: The present invention relates to a cytotoxic antibody conjugate having excellent cytotoxic and antitumor properties, which is represented by the formula:

Also, a general method for producing a cytocidal antibody conjugate containing the above, i.e., _a method for producing a cytocidal antibody conjugate containing the above, comprising: a halogenated acetylhydrazide derivative of a cytocidal substance represented by the general formula [II
I〕 (RCONHNHCOCH ₂ NH _n Ab ………〔III〕 The present invention also provides a method for producing a cytotoxic antibody complex represented by the formula:

In the present invention, RCO in the general formula [II] and [III]
is represented by the following general formula [IV] In the case where the antibody or a fragment thereof is represented by the following general formula [II'] The present invention also includes a method for producing the cytocidal antibody conjugate represented by the formula [I], which comprises reacting a cytocidal antibody conjugate represented by the formula [I] with a halogenated acetylhydrazide of a folate-antagonistic folate analogue represented by the formula [I].

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, an antibody refers to an immunoglobulin obtained by selectively binding to a specific antigen possessed by cells to be killed (hereinafter referred to as target cells). Such antibodies are produced, for example, as follows: Antisera isolated from animals such as monkeys, horses, cows, goats, sheep, rabbits, and chickens immunized with tumor cells or specific target cells such as lymphocytes are subjected to ethanol fractionation, ammonium sulfate fractionation, ion exchange or molecular sieve column chromatography, protein A fractionation, and the like.
The antibodies used in the present invention can be obtained from the culture medium of transformed cells obtained by virus-transforming lymphocytes collected from an animal immunized with target cells, or from specific antibody-producing fused cells (hybridomas) obtained by cell fusion of these lymphocytes with myeloma cells or the like, or from the serum or peritoneal fluid of an animal after inoculating these cells. In addition to target cells, antigens extracted from target cells or antigens of artificially synthesized target cells can also be used as antigens for immunization to obtain the above-mentioned antisera or antibody-producing lymphocytes. Furthermore, the antibody-producing lymphocytes used for transformation or cell fusion in the production of the above-mentioned antibodies can be:
Lymphocytes obtained from humans and sensitized to antigens, if necessary treated with antigenic substances, can also be used.
There are known to be five types: IgG, IgA, IgM, IgD, and IgE, and any of these can be used in the present invention.

In the present invention, antibodies can be used either as they are or as fragments thereof (eg, Fab, Fab', F(ab') ₂ ) as long as they contain a portion capable of binding to an antigen.

In the present invention, antibodies also include those that have been chemically modified, for example, by acylation, as necessary.

The cytotoxic antibody conjugate of the present invention generally comprises an antibody or a fragment thereof represented by the general formula [II]: The compound can be obtained by reacting the compound represented by the formula (I) with a halogenated acetylhydrazide derivative of a cytocidal substance represented by the formula (I).

In the general formula [II], R is CO
The carboxyl group, when taken together with the carboxyl group, represents a monovalent organic group derived from a cytotoxic substance, and a wide range of substances can be used as such cytotoxic substances, as long as they originally have a carboxyl group or have been derived into a derivative having a carboxyl group. Examples of such cytotoxic substances that can be suitably used include the following substances:

Nitrosourea derivatives Chlorambucil Mitomycin-C derivatives 5-fluoro-2'-deoxyuridine derivatives Desacetylvinblastine derivatives In the present invention, a cytocidal substance that is particularly preferably used is a folate-antagonistic folic acid analog. A folate-antagonistic folic acid analog is a substance that antagonistically inhibits the action of folic acid in cells, and has the following structure of folic acid: It refers to a substance having a carboxyl group with a structure similar to that of the compound shown in Table 1. Examples of such substances include methotrexate, aminopterin, and 10-ethyl-10-deazaaminopterin.

In the general formula [I], R' is C
Together with O, it represents the residue of an antifolate folic acid analog.
Examples of such organic groups include: (1) methotrexate residues; and (2) aminopterin residue and (3) 10-ethyl-10-deazaaminopterin residue and The following can be mentioned:

The oligopeptide used in the present invention includes those having 3 to 6 amino acid residues. Suitable examples thereof include L-leucyl-L-alanyl-L-leucine, L-leucyl-L-alanyl-L-leucyl-L-alanine, L-alanyl-L-leucyl-
Other examples include glycylglycylglycine, glycyl-L-phenylalanine-L-leucine,
Examples include glycyl-L-phenylalanyl-L-phenylalanine-L-leucine.

In the general formula (II or II'), the halogen atom represented by X is preferably an iodine, bromine or chlorine atom, with an iodine atom and a bromine atom being particularly preferred.

In the present invention, L-leucyl-L-alanyl-
The oligopeptide containing L-leucine is not particularly limited as long as it does not prevent the intracellular cleavage of L-leucyl-L-alanyl-L-leucine.
When the oligopeptide contains amino acids other than L-leucine and L-alanine, it is preferable that such amino acids do not have functional groups with high chemical reactivity. Particularly preferred oligopeptides containing L-leucyl-L-alanyl-L-leucine are L-leucyl-L-alanyl-L-
Examples include leucine, L-leucyl-L-alanyl-L-leucyl-L-alanine, and L-alanyl-L-leucyl-L-alanyl-L-leucine.

In addition, the general formulas [II] and [IV] used in the present invention
Antifolate folate analog oligopeptide represented by
The halogenated acetylhydrazide derivatives include, for example, oligopeptides of antifolate folic acid analogs represented by the following formula: R'-CO-NH-Y-CO- _OCH3 (where R' and Y are defined as in formula [IV]).
The ester derivative can be treated with hydrazine hydrate to form a hydrazide derivative, which is then reacted with an activated ester of a halogenated acetic acid.

In the method of the present invention, it is preferable to use 1 to 100 moles of the oligopeptide halogenated acetylhydrazide derivatives of the antifolate folate analogs represented by formulas [II] and [IV] per mole of the antibody or its fragment,
The reaction may be carried out, for example, at pH 6 to 10 for the antibody or fragment thereof.
9 buffer solution (protein concentration is preferably 1 to 40 mg/
To a solution (adjusted to a total volume of 1 ml) is added a halogenated acetylhydrazide derivative of an antifolate oligopeptide analog of folic acid dissolved in a small amount of solvent, such as N,N-dimethylformamide, with stirring at 0 to 37°C, and the reaction is continued for 15 minutes to 24 hours. The reaction mixture is then subjected to gel filtration or dialysis to remove unreacted halogenated acetylhydrazide derivative of an antifolate oligopeptide analog of folic acid, thereby purifying the cytocidal antibody conjugate represented by formula [I].

The present invention will be described in detail below with reference to examples.

Example 1 (a) Production of cytotoxic antibody conjugate Mouse monoclonal antibody against mouse breast cancer cell MM46
9.1 mg of IgG2a was dissolved in 0.1 M sodium chloride.
Dissolve in 1.1 ml of 1 M Tris-HCl buffer (pH 8.0),
To this, the following formula [II-1]: and a methotrexate (hereinafter referred to as MTX) derivative represented by the following formula [II-2]: [The definition of A is the same as in formula [II-1].]
50 μL of an N,N-dimethylformamide solution (48.8 mg/ml) of the X derivative mixture was added, and the mixture was allowed to react at room temperature for 17 hours. After the reaction, phosphate buffered saline (hereinafter referred to as PB
The resulting solution was thoroughly dialyzed against a cytotoxic antibody complex (hereinafter referred to as "MM46 antibody complex") to obtain 6.8 mg of a cytotoxic antibody complex containing an average of 9.4 MTX derivatives bound to one anti-MM46 antibody molecule.
The absorbance at 80 nm and ₃₇₂ nm ( _referred to as A and A, respectively) was measured to determine the MTX concentration. The IgG concentration was calculated by correcting _A with _the MTX concentration, and the average number of MTX molecules bound per IgG molecule was calculated.

The MTX derivative is prepared by first adding a peptide, L-leucyl-L-alanyl-L-leucyl-L-alanine methyl ester, synthesized by a conventional peptide synthesis method to MTX and condensing it with a condensing agent (N,N'-dicyclohexylcarbodiimide).
and converting the methyl ester of the resulting condensed peptide into a hydrazide using hydrazine hydrate.
The hydrazine residue was then iodoacetylated with paranitrophenyl iodoacetic acid to give the desired hydrazine moiety.

(b) Cytocidal effect of cytotoxic antibody conjugates on cultured tumor cells. Mouse breast cancer MM46 cells were suspended in RPMI 1640 medium containing 10% fetal bovine serum at a concentration of 2.5 x ¹⁰ cells/ml, and 0.2 ml of each was dispensed into each well of a 96-well flat-bottom plate. Next, 0.02 ml of various concentrations of the cytotoxic antibody conjugate (prepared in (a) above) dissolved in the medium was added, stirred, and cultured at 37°C in a 5% _CO2 atmosphere for 3 days. The above procedures were performed aseptically. After culture, 0.02 ml of a 3% trypan blue solution was added, and viable cells not stained by trypan blue were counted under a microscope.

The results are summarized in Table 1. When nothing was added, MM46 cells proliferated to approximately 1 x 10 ⁶ /ml after 3 days of culture, but when the cytocidal antibody complex prepared in (a) was added, the number of viable cells decreased depending on the amount added.

In particular, at 10 μM equivalent to MTX, the number of viable cells was 1% or less compared to when the cells were cultured without the addition of the antibody complex, demonstrating a very high cell-killing effect.

(c) Proof that the iodoacetylhydrazide derivative of methotrexate reacts with the amino group of an antibody (immunoglobulin) molecule. The N,N of the iodoacetylhydrazide derivative of methotrexate (MTX derivative) represented by the above formula [II-1]
-Dimethylformamide solution (13.4 mg/ml) 100
μ represents rabbit gamma globulin (hereinafter referred to as RGG);
solution (5 mg/ml, 0.1 M sodium chloride
Dissolved in M Tris-HCl buffer (pH 8.0) 1 ml
In one experiment, 100 μl of 1 M ethanolamine was added to the RGG solution in advance, and in another experiment, 100 μl of 0.9% sodium chloride was added to the RGG solution in advance.

After reacting at room temperature for 14 hours, the reaction mixture was diluted with 10 M phosphate buffer (pH 7.2) containing 0.14 M sodium chloride.
(hereinafter referred to as PBS) to remove low molecular weight reagents. The dialyzed solution was centrifuged, and 280
The absorbance at 372 nm and 372 nm was measured. The MTX concentration was calculated from _{A 372} and A ₂₈₀ was corrected by the MTX absorption to obtain R
Thus, the reaction rate of the MTX derivative and the binding ratio of MTX to RGG were determined.

The results are shown in Table 2. Under the above conditions, an average of 5.8 MTX derivatives bound to one RGG molecule without the addition of ethanolamine. However, when ethanolamine was added, the reaction was significantly suppressed to 0.6. This demonstrated that the reaction between the MTX derivatives and RGG was mediated by the amino group.

Example 2 Comparison of the cytotoxicity of anti-MM46 cytotoxic antibody conjugate, nonspecific globulin conjugate, and MTX derivative before globulin binding. Using normal (nonspecific) mouse γ-globulin instead of anti-MM46, a nonspecific globulin conjugate was obtained in which an average of 7.9 MTX derivatives described in Example 1 were bound per globulin molecule by a method similar to that of Example 1(a).

The anti-MM46 cytotoxic antibody conjugate, non-specific globulin conjugate, and M before globulin binding prepared in Example 1(a) were
The TX derivatives were cultured in MM46 as in Example 1(b).
The cytocidal effect on cells was examined.

The results are summarized in Table 3. The anti-MM46 cytotoxic antibody conjugate exerted its cytotoxic effect at a much lower concentration than the other two. That is, the concentration of antibody conjugate (equivalent to MTX) required to suppress the number of viable L1210 cells to 50% of that observed when cultured without the antibody conjugate was approximately one-tenth that of the nonspecific conjugate.

Furthermore, the difference in activity between the anti-MM46 conjugate prepared by the method of the present invention in which MTX is conjugated via a tetrapeptide and the corresponding non-specific conjugate was compared with the difference in activity between the anti-MM46 conjugate prepared by the conventional method in which MTX is directly conjugated using an active ester of MTX and the corresponding non-specific conjugate.
The former was four times larger.

Example 3: Preparation of anti-human melanoma cytotoxic antibody conjugate and evaluation of its antitumor activity. Using a mouse monoclonal antibody IgG2a against human melanoma cells and normal (non-specific) rabbit γ-globulin instead of anti-MM46 antibody, the MTX derivative described in Example 1 was conjugated to IgG1 in the same manner as in Example 1(a).
The anti-human melanoma cytocidal antibody conjugate and the nonspecific globulin conjugate were obtained, each containing 5.4 and 4.4 molecules per molecule, respectively. These conjugates were used in the antitumor evaluation test described below.

Nude mice (5 mice per group) were subcutaneously transplanted with human melanoma cells KHm-1 in the ventral region, and the mice were then treated with 500 mg/kg of steroids from day 10 of tumor transplantation.
Anti-human melanoma cytotoxic antibody conjugate, 3 times every other day;
A non-specific globulin conjugate and a mixture of anti-human melanoma antibody and MTX in the same amount as contained in the anti-human melanoma cytocidal antibody conjugate were administered.

Nude mice (10 mice) that were not administered with the test sample served as a control group. On the 30th day after tumor transplantation, the tumors were excised and their weights were measured. Only in the group administered with the anti-human melanoma cytotoxic anti-body complex,
A significant inhibition of tumor growth was observed, with the average weight being 27% of that of the control group.

Example 4 Instead of the mixture of the MTX derivative represented by the formula [II-1] and the MTX derivative represented by the formula [II-2] in Example 1, a mixture of the MTX derivative represented by the following formula [II-3], [A is defined as in formula [II-1].] and an aminopterin (hereinafter referred to as AMN) derivative represented by the following formula [II-4]: [The definition of A is the same as in formula [II-1].]
By the same method as in (a), an anti-MM46 cytotoxic antibody complex was prepared in which 7.2 AMN derivatives were bound to one IgG molecule.

Example 5 Instead of the mixture of MTX derivatives represented by formula [II-1] in Example 1, a mixture of MTX derivatives represented by the following formula [II-5]: and an MTX derivative represented by the following formula [II-6]: [E is defined as in formula [II-5].]
By the same method as in (a), an anti-MM46 cytotoxic antibody complex was prepared in which 5.7 MTX derivatives were bound to one IgG molecule.

Example 6 Instead of the anti-MM46 antibody in Example 1, the anti-human melanoma antibody used in Example 3 was used, and the MTX derivative represented by formula [II-1] and the MTX derivative represented by formula [II-2] were used.
Instead of a mixture of derivatives, the compound of the following formula [II-7]: [E is defined as in formula [II-5].] and an AMN derivative represented by the following formula [II-8]: [E is defined as in formula [II-5].]
By the same method as in (a), an anti-human melanoma cytotoxic antibody complex was prepared in which 6.1 AMN derivatives were bound to one IgG molecule.

Example 7 Instead of the mixture of the MTX derivative represented by the formula [II-1] and the MTX derivative represented by the formula [II-2] in Example 1, a mixture of the MTX derivative represented by the following formula [II-9]: and an MTX derivative represented by the following formula [II-10]: [The definition of G is the same as in formula [II-9].]
By the same method as in (a), an anti-MM46 cytotoxic antibody complex was prepared in which 8.2 MTX derivatives were bound to one IgG molecule.

Example 8 Instead of the mixture of the MTX derivative represented by the formula [II-1] and the MTX derivative represented by the formula [II-2] in Example 1, a mixture of the MTX derivative represented by the following formula [II-11], [A is defined as in formula [II-1].] and a 10-ethyl-10-deazaaminopterin (hereinafter referred to as 10-EdAM) derivative represented by the following formula [II-
12], [The definition of A is the same as in formula [II-11].] An anti-human melanoma cytocidal antibody complex was prepared in the same manner as in Example 1(a) using a mixture of 10-EdAM derivatives represented by the formula:

Example 9 Instead of the mixture of the MTX derivative represented by the formula [II-1] and the MTX derivative represented by the formula [II-2] in Example 1, a mixture of the MTX derivative represented by the following formula [II-13], [E is defined as in formula [II-5].] and an AMN derivative represented by the following formula [II-14]: [E is defined as in formula [II-5].]
By the same method as in (a), an anti-MM46 cytotoxic antibody complex was prepared in which 2.2 AMN derivatives were bound to one IgG molecule.

──────────────────────────────────────────────────── Continued from the front page (56) References: JP 51-126281 (JP, A) JP 59-186924 (JP, A) JP 61-501449 (JP, A) Chemical & Pharmaceutical Bulletin Vol. 35, No. 3, pp. 1128-1137 (Published March 1987)

Claims (5)

[Claims] 1. A cytotoxic antibody complex represented by the above general formula [I] 2. The cytotoxic antibody complex according to claim 1, wherein the antibody is a monoclonal antibody. 3. The oligopeptide is L-leucyl-L-alanyl-L-leucyl-L-alanine, L-alanyl-L
-Leucyl-L-alanyl-L-leucine, or L-
3. The cytocidal antibody conjugate of claim 1 or 2, which is leucyl-L-alanyl-L-leucine. 4. The cytotoxic antibody conjugate of any one of claims 1 to 3, wherein the folate-antagonistic folate analog is methotrexate, aminopterin, or 10-ethyl-10-deazaaminopterin. 5. The antibody or fragment thereof, wherein the antibody or fragment thereof is a compound represented by the following general formula [II']: with a halogenated acetylhydrazide of a folate antagonistic folic acid analogue represented by the following general formula [I]: A method for producing a cytotoxic antibody complex represented by the formula: