JPH0428720B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel selective cell-killing protein complex and a method for producing the same. For more information, see Antitumor IgG
The present invention relates to a novel selective cell-killing protein complex having a component consisting of an immunoglobulin or a fragment thereof containing an antigen-binding site and a component consisting of the anticancer drug neocarzinostatin, and a method for producing the same. For the purpose of selectively killing only certain types of cells, attempts have been made to combine various anticancer drugs with immunoglobulins that can selectively bind to the target cells (for example, T. Ghose).
et al., Journal of the National Cancer Inst.
61, pp. 657-676, 1978). Neocarzinostatin is considered to be suitable as an anticancer agent for such purposes because of its strong cytotoxicity, and a complex with immunoglobulin was investigated. For example, Kimura et al.
WSCI) was used as a dehydration condensation agent to bind immunoglobulin and neocarzinostatin, and the resulting complex possessed antibody activity as well as neocarzinostatin activity, and was found to be more effective than neocarzinostatin alone. effectively target cells
It has been reported that it inhibited DNA synthesis (see Cancer and Chemotherapy, Vol. 6 Extra Edition, pp. 75-81, 1979). In addition, G. Jung et al. created a complex by forming a disulfide bond between the sulfur atoms introduced into immunoglobulin and neocarzinostatin using a cross-linking agent, and the complex was developed into neocarzinostatin. It has been reported that nostatin activity was maintained (Biochemical and Biophysical Research Co., Ltd.).
101, pp. 599-606, 1981). However, when preparing the complex, WSCI
In the method of using WSCI as a condensing agent, WSCI is a reagent that causes dehydration condensation between an amino group and a carboxyl group to form an amide bond. Cross-linking between immunoglobulins, cross-linking between neocarzinostatins, and cross-linking between neocarzinostatins also occur, making it difficult to obtain desired products. In addition, since immunoglobulin and neocarzinostatin have multiple amino groups and carboxyl groups, a considerable amount of polymeric substances in which multiple immunoglobulins and neocarzinostatin are bonded to each other is produced, and cross-linking using WSCI is required. Depending on the method, it is extremely difficult to obtain a complex that can actually be used for treatment. In addition, in the above-mentioned complex of Jung et al., immunoglobulin and neocarzinostatin are disulfide (-S-S
-) bonds, and since a significant portion of such unnatural disulfide bonds are cleaved in the blood, such complexes have the disadvantage that their efficacy is reduced before they reach the target cells. The present inventors have conducted extensive research in order to overcome these drawbacks of the prior art and develop a selective cell-killing protein complex that selectively exhibits strong toxicity to cells to be killed, such as cancer cells. As a result, the present invention was achieved. That is, the present invention provides the following formula [] which is obtained by binding an anti-tumor IgG immunoglobulin or a fragment thereof to neocarzinostatin. [wherein Ab represents an antitumor IgG immunoglobulin or a fragment thereof, and Ab is bonded through an amino group in its chemical structure. NCS stands for neocarzinostatin, and NCS is attached through an amino group in its chemical structure. X represents an m-phenylene group or a trimethylene group, and m represents 0 or 1. S represents a sulfur atom, and when m is 0, S is derived from a thiol group originally contained in the chemical structure of the antitumor IgG immunoglobulin or its fragment;
When m is 1, it originates from a thiol group contained in the introduced organic group. n represents an integer of 1 to 5. ] It is a selective cell-killing protein complex represented by In the present invention, immunoglobulin refers to an antibody that recognizes a target cell to be destroyed or a globulin containing such an antibody. Such immunoglobulin is
When the target cell is a cancer cell, it is called an antitumor immunoglobulin. Anti-tumor immunoglobulins can be obtained, for example, from the serum of cancer patients, or from tumor cells or tumor-specific or tumor-associated antigens from monkeys, horses, cows, etc.
Cohn's ethanol fractionation method, ammonium sulfate fractionation method,
Proteins with antibody activity (immunoglobulins) that can be prepared by known means such as ion-exchange chromatography and, if necessary, subjected to absorption and adsorption operations using various cells. Alternatively, antibody-producing lymphocytes obtained by immunizing an animal with cancer cells or cancer antigens are fused with, for example, bone marrow tumor cells to obtain culturable antibody-producing fusion cells (hybridomas), which are then inoculated into
An immune system with highly specific antibody activity that is prepared from culture fluid, serum, or peritoneal fluid after being cultured in vitro (externally) or transplanted into animals and cultured in vivo (in vivo). Contains globulin. Furthermore, the immunoglobulin of the present invention also includes an immunoglobulin having antibody activity that is prepared by releasing anti-tumor antibodies from tumor tissue using a denaturing agent such as a surfactant, and then preparing the anti-tumor antibody therefrom by the same means as described above. Immunoglobulins include IgG, IgA, IgM, IgD,
It is known that there are five classes of IgE, and their basic structure, as schematically shown in Figure 1, consists of two L chains, indicated by L, and an H chain, indicated by H.
Two chains have at least three disulfide bonds (-
S-S-bond), and
As shown in Figure 1, they are identical in that they consist of a Fab portion with antigen-binding activity and an Fc portion with effector activity. The immunoglobulin used in the present invention is an IgG class immunoglobulin among the above five classes. Anti-tumor IgG immunoglobulin or a fragment thereof is used as one component of the selective cell-killing protein complex of the present invention, and the fragment of anti-tumor IgG immunoglobulin has the antibody activity of the above-mentioned immunoglobulin. A fragment containing the site is used. Such fragments include, in particular, Fab, Fab' (a portion consisting of a Fab part and a so-called hinge part (the shaded part in Figure 1B)), and F(ab)' ₂ , which is a dimer of Fab'. desirable. Neocarzinostatin used in the present invention is a protein antibiotic with a molecular weight of approximately 11,000 obtained from the culture solution of Streptomyces Carzinostaticus that exhibits antitumor activity against acute leukemia, gastric cancer, bladder cancer, liver cancer, etc. It has two free amino groups. In addition, neocarzinostatin is dependent on a non-protein part, that is, a chromophore, and this chromophore is responsible for the biological activity of neocarzinostatin, and the protein part is involved in the stabilization, transport, activation, etc. of the chromophore. (Edo, Ishida, History of Medicine, Vol. 120, 79-80)
1982), such molecular structural features are advantageously used in the present invention. In the above formula [], when m=0, S is a sulfur atom derived from an immunoglobulin or a fragment thereof, and when m=1, it is a sulfur atom introduced by a crosslinking agent. In the formula [],
For example, the dimethylene group has the following formula [] [In the formula, Y is a monovalent organic group that can form an active disulfide group together with the bonded sulfur atom S,
X ₃ represents a dimethylene group, and Z represents an alcohol residue of an active ester. ] Derived from the crosslinking agent represented by Specific examples of the monovalent organic group represented by Y that can form an active disulfide group together with the bonded sulfur atom include 2-pyridylthio group.

[Formula] 4-pyridylthio group

[Formula] 3-carboxy-4-nito lophenylthio group

[Formula] 4-carboxy-2-pyridyldithio group

[Formula] N-oxy-2-pi lysyldithio

[Formula] 2-nitrophe Nylthio group

[Formula] 4-nitro-2- Pyridylthio group

[Formula] 2-be nzothiazoylthio group

[Formula] 2-benzimidazoylthio group

[Formula] N-phenylamino- N′-phenyliminomethylthio group

[Formula] etc. can be mentioned. A specific example of the alcohol residue of the active ester represented by Z is an N-succinimidoxy group.

[Formula] N-hydroxy-5-norbornene-2,3-dicarbodiimidoxy group

[Formula] N-phthalimidoxy base

[Formula] p-nitrophenoxy group, 2,4-dinitrophenoxy group, 2,4,
Examples include 5-lolichlorophenoxy group and bentachlorophenoxy group. Specific examples of the alcohol residue of the imidoester represented by Q include methoxy and ethoxy groups. Specific examples of the halogen atom represented by R include chlorine, bromine, and iodine. X in the above formula [] is the following formula [] [In the formula, X ₄ represents a m-phenylene group or a trimethylene group. The definition of Z is the same as the formula []. ] Derived from X ₄ of the crosslinking agent represented by Specific examples of the alcohol residue of the active ester represented by Z are the same as in the case of the above formula []. Specific examples of the crosslinking agent include N-succinimidyl 3-(2-pyridyldithio)propionate as a crosslinking agent represented by the formula [], and N-hydroxysuccinimide benzoate as a crosslinking agent represented by the formula []. Imidoester and maleimidoacetic acid 2,4-dinitrophenol ester can be mentioned. The selective cell-killing protein complex of the present invention has anti-tumor properties.
By generating or introducing a thiol group into IgG immunoglobulin or its fragment, the following formula [] [wherein Ab represents an antitumor IgG immunoglobulin or a fragment thereof, and Ab is bonded through an amino group in its chemical structure. m represents 0 or 1. S represents a sulfur atom, but when m is 0, S
m is derived from a thiol group originally contained in the chemical structure of the antitumor IgG immunoglobulin or its fragment, and when m is 1, it is derived from a thiol group contained in the introduced organic group. n1 represents an integer from 1 to 5. ] A compound represented by is synthesized, and on the other hand, the following formula [] [In the formula, NCS represents neocarzinostatin,
NCS is bonded through amino groups in its chemical structure. X represents m-phenylene group or trimethylene group] It can be produced by synthesizing neocarzinostatin having an introduced maleimide group represented by the following and reacting the two. That is, anti-tumor IgG immunoglobulin or a fragment thereof is reacted with, for example, a crosslinking agent represented by the formula [], and the product is reduced with, for example, 2-mercaptoethanol or dithiothreitol [reaction (1)]. , an immunoglobulin or a fragment thereof into which a thiol group represented by formula [X] has been introduced is obtained. (In the above formula (1), p represents an integer of 1 to 5.) On the other hand, neocarzinostatin is expressed by the formula []
It is reacted with a crosslinking agent represented by the following formula [X
] [In the formula, the definitions of NCS and X are the same as in the case of the formula []. q represents 1 or 2. ] Obtain neocarcinostatin into which a maleimide group represented by the following is introduced. Then, the selective cell-killing protein complex of the present invention is produced by reacting the thus obtained antitumor IgG immunoglobulin or its fragment into which a thiol group has been introduced with neocarzinostatin into which a maleimide group has been introduced. can be manufactured. One precursor of the selective cell-killing protein complex of the present invention is an immunoglobulin or a fragment thereof having a thiol group; In addition, if the immunoglobulin or its fragment itself originally has a thiol group, or if it has a cystine-based disulfide bond, the disulfide group can be reduced, for example. It may also be a thiol group that can be In the production of the selective cell-killing protein complex of the present invention, when reacting an immunoglobulin or its fragment with a crosslinking agent, 1 to 1 to 10% of the crosslinking agent is added to 1 mole of the immunoglobulin or its fragment.
Preferably, 100 mol is used. The reaction is carried out using an immunoglobulin or its fragment at a protein concentration of 0.5 to 100 mg/ml (more preferably 1
~20mg/ml) in a solution prepared with 0
An aqueous solution of the crosslinking agent or, if the crosslinking agent is not soluble in water, a small amount of an organic solvent such as N,N-dimethylholdamide,
It is carried out by adding a solution dissolved in dimethyl sulfoxide, 1,2-dimethoxyethane, methanol, ethanol, acetone, etc. The reaction time depends on the reaction scale and reaction conditions, but is generally within 2 days. After the reaction is completed, unreacted crosslinking agents and low-molecular reaction products are removed by dialysis or molecular sieve column chromatography. When a disulfide group is introduced by a crosslinking agent, the disulfide group is reduced to a thiol group, but this reaction uses an excess of a thiol reagent (e.g., 2-mercaptoethal, dithiothreitol) and the above reaction temperature. , reaction time can be applied, and the reactant can be purified by the same method as described above. Furthermore, the reaction conditions for introducing a maleimide group into neocarzinostatin using a crosslinking agent are the same as those for reacting the crosslinking agent with the above-mentioned immunoglobulin or its fragment. The reaction between an immunoglobulin having a thiol group or a fragment thereof having a thiol group generated or introduced therein and neocarzinostatin having a maleimide group introduced can be carried out by mixing the two (the preferred pH of the reaction solution is 5 to 8, The preferred protein concentration is 1-20mg/
ml) at 0-40°C for 2-24 hours. The complex of immunoglobulin or its fragment and neocarzinostatin obtained by the above method can be separated and purified from the reaction mixture by a commonly used procedure, such as molecular sieve column chromatography. can. The selective cell-killing protein complex of the present invention has a component consisting of neocarzinostatin that is toxic to target cells such as cancer cells, and a component that selectively recognizes target cells and targets neocarzinostatin. It has a component consisting of immunoglobulin or its fragment, which is a carrier that selectively reaches cells, and both components are linked by a chemically safe bond, so it is selective for cytotoxicity against target cells. It has the characteristic of being able to perform effectively and efficiently. Moreover, such a complex can be produced with high purity by the method of the present invention. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 (a) Preparation of anti-mouse breast cancer MM46 monoclonal antibody Anti-MM46 IgG2b antibody-producing hybridoma obtained by cell fusion method (Seto Kadai et al., Journal of Immunology (J. Immunol), Vol. 128,
201-205, 1982) was inoculated into the peritoneal cavity of 15 nude mice at 2 x 10 ⁷ per mouse, and after 10 days, the ascites fluid was collected.
It was thoroughly dialyzed against 0.1M phosphate buffer (PH8.0). The dialysis solution was thoroughly equilibrated with the same buffer using a Protein A/Sepharose column (column size 1.5).
x 12.5cm) to thoroughly wash out the protein that passed through, elute the impure protein with 0.1M citrate buffer (PH5.0), and then elute the impure protein with 0.1M citrate buffer (PH5.0).
3.0) to elute the adsorbed IgG2b and collect the eluate.
Return the pH to neutral with 2MTris-HC buffer (PH8.2), then add 20mM phosphate buffer (PH7.5) in step 5.
After thorough dialysis, 105 mg (17.7 ml) of anti-MM46 monoclonal antibody (IgG2b) was obtained. In addition, from 50 ml of normal mouse serum, we collected IgG2b (hereinafter referred to as "non-immune") that does not have affinity for MM46 in the same manner as above.
25 mg (7.0 ml) of IgG2b was obtained. (b) Preparation of IgG2b antibody with thiol group Anti-MM46 monoclonal antibody IgG2b27.3 obtained in (a) above
36.4 μl of a 20 mM ethanol solution (4 times equivalent of IgG2b) of N-succinimidyl 3-(2-pyridyldithio)propionate (hereinafter abbreviated as SPDP) was added to mg (4.6 ml, 0.182 μmole), and
After reacting for minutes, add 2MTris-HC to the reaction solution.
1/10 volume of buffer solution (PH7.5), 2M2-ME
10 μl (30 times equivalent to SPDP) was added and incubated at 37° C. for 1 hour. Then, the reaction solution was diluted with 20mM phosphate buffer (PH7.5).
Approximately 20 mg of
(4.76ml) of anti-thiol group introduced
MM46IgG2b antibody was obtained. Similarly, for non-immune Ig2b, approximately 20 mg (7.9
ml) of IgG2b into which a thiol group was introduced was obtained. (c) Preparation of m-maleimidobenzoyl neocarzinostatin N-succinimidyl m - N of maleimidobenzoate (hereinafter abbreviated as SMB),
N′-dimethylformamide solution (173mM) 0.15
ml was added and reacted at 23°C for 30 minutes. The reaction solution was centrifuged and the supernatant was transferred to a Sephadex G-25 column (1 cm) equilibrated with 50 mM phosphate buffer (PH6.2).
x 36 cm), remove unreacted low-molecular substances such as SMB, and prepare 9.9 mg (4.5 ml) of m-maleimidobenzoyl neocarcinostatin (hereinafter abbreviated as NCS-MB).
I got it. In addition, 10 times the equivalent of dinitrophenyl-cysteine (hereinafter abbreviated as DNP-cysteine) was added to 100 μl of a portion of the product, incubated at 23°C for 1 hour, and transferred to Cephadex G-25 equilibrated with physiological saline. After removing unreacted DNP-cysteine, the absorbance of the resulting sample at 278 nm and 361 nm was measured (NCSE ¹ % ₁ cm, 278 nm = 15, DNP
- Cysteine E% ₁ cm, 361 nm = 17000), on average
It was found that 0.32 m-maleimidobenzoyl groups were introduced per NCS. (d) Preparation of a complex of anti-MM46IgG2b antibody and NCS 20mg (4.76ml) of the anti-MM46IgG2b antibody into which a thiol group was introduced as obtained in (b) above was added to the above (c).
4.73 mg (2.15 ml) of NCS-MB obtained as follows.
Add sodium hydroxide to adjust the pH to 7.0 at 4℃.
The mixture was allowed to react overnight. After the reaction was completed, the reaction solution was applied to a Cephadex G-150 Superfine column (1.5 x 89 cm) equilibrated with physiological saline, and the fractions shown in the shaded area in Figure 2 were collected and concentrated to obtain the complex of the present invention. 20.8 ml (6.1 ml) of containing product were obtained. When the obtained product was analyzed by sodium lauryl sulfate polyacrylamide gel electrophoresis (hereinafter abbreviated as SDS-PAGE), disk 2 in Figure 3 was found.
A band pattern as shown in Figure 1 was obtained, and this product was composed of unreacted anti-MM46 IgG2b antibody (molecular weight approx.
155,000), an IgG2b antibody bound to one NCS (molecular weight approximately 170,000), and an IgG2b antibody bound to two NCS (molecular weight approximately 180,000). Same as above using non-immune IgG2b antibody.
When the reaction with NCS was carried out, the product was
The analysis results by SDS-PAGE were as shown in disk 3 of FIG. 3, and were similar to those for the anti-MM46 IgG2b antibody. Note that disk 1 in FIG. 1 is the band pattern of anti-MM46 IgG2b antibody. (e) Therapeutic experiment in mice The product containing the complex of anti-MM46IgG2b antibody and NCS prepared as described in (d) above (hereinafter simply referred to as anti-MM46 IgG2b antibody and NCS)
MM46IgG2b antibody and NCS complex)
We investigated the therapeutic effects on mice transplanted with MM46 tumors. That is, 3 × 10 ⁵ mice were administered to each group of 5 C3H mice.
MM46 cells were transplanted intraperitoneally, and 24 hours after transplantation, samples (complex of anti-MM46 IgG2b antibody and NCS, complex of non-immune IgG2b antibody and NCS, anti-MM46 IgG2b antibody and NCS,
50μg of 1:1 mixture of MM46IgG2b antibody and NCS
The lifespan of mice in each group was compared, and the antitumor activity of the complex of anti-MM46 IgG2b antibody and NCS was examined. The results are shown in Figure 4. The conjugate of non-immune IgG2b antibody and NCS showed no antitumor activity even when administered at 500 μg, and the lifespan of the mice was similar to that of the saline-treated control group, with all mice dying by 15 days after tumor implantation. (Figure 4(b)). Anti-MM46IgG2b antibody and NCS
A 1:1 mixture of 500 and 50 μg showed some antitumor effects, but only one mouse survived for a long period of time (Figure 4(c)). On the other hand, in the group administered with the anti-MM46IgG2b antibody and NCS complex, three mice survived for a long period of time when 500 μg was administered, indicating that the complex of the present invention has strong antitumor properties (Figure 4). (a)). Example 2 (a) Preparation of anti-mouse leukemia L1210 IgG 1×10 ⁶ mouse leukemia L1210 cells were made into an emulsion with Freund's complete adjuvant and intravenously injected into rabbits. Thereafter, approximately 1×10 ⁶ L1210 cells were subcutaneously injected together with adjuband three times at one-week intervals, and blood was collected 8 days after the final administration. The obtained blood was pooled, the serum was separated, and the serum was inactivated by heating at 56°C for 30 minutes. To 200 ml of the anti-L1210 serum obtained in this way,
200 ml of a saturated aqueous solution of ammonium sulfate was added, and the resulting precipitate was separated by centrifugation. This precipitate
Dissolve in 50ml of 0.01M phosphate buffer (PH7.6),
Furthermore, it was thoroughly dialyzed against the same buffer. This dialyzed solution was subjected to DEAE cellulose column chromatography (column size 3 cm x 94 cm) equilibrated with the same buffer, and the unadsorbed fraction was treated with anti-antibiotics.
A solution containing L1210IgG was obtained. (b) Separation of F(ab)′ ₂ fragment from immunoglobulin 1.2 g of anti-L1210 IgG obtained as in (b) above
Dissolve in 40ml of 0.1M acetate buffer (PH4.5) and add 24mg
After adding pepsin and decomposing at 37°C for about 18 hours, the decomposition products were separated into physiological saline.
G200 column chromatography (column size
3.5 cm x 140 cm), a pure F(ab)' ₂ fragment was obtained as a protein flowing out at a molecular weight of approximately 100,000. (c) Preparation of Fab' fragment 2.0ml of 0.01M Tris-HCl-0.14M sodium chloride-2mMEDTA solution (PH8.3) containing 18.4mg of F(ab)' ₂ fragment obtained as in (b) above.
0.02ml of 150mM 2-mercaptoethanol aqueous solution was added to the mixture, and the mixture was reduced at 37°C for 1 hour. After the reaction, the solution was mixed with 5mM acetate buffer - 0.14M sodium chloride - 1mM MEDTA solution (PH5.5) (hereinafter referred to as
Sephadex G25 column chromatography (1.0cm x 20cm) equilibrated with ANE buffer)
2-mercaptoethanol was removed to obtain a Fab' fragment having one thiol group. (d) Anti-mouse leukemia L1210 IgG fragment
Preparation of complex of Fab′ and NCS The anti-L1210 IgG obtained as in (c) above
5 mg (2 ml) of the Fab' fragment was added to the Fab' fragment of Example 1.
3.5 mg (1.6 ml) of NCS-MB obtained as in (c)
In addition, 1/10 volume of 0.5M phosphate buffer (PH7.0) was added, and the mixture was reacted overnight at 4°C. After the reaction is complete, Cephadex G- is equilibrated with physiological saline.
The complex was purified using a 150 Superfine column (1.5 cm x 89 cm) to obtain 6 mg (4 ml) of a complex of anti-L1210 IgG Fab' fragment and NCS. When the obtained complex was analyzed by SDS-PAGE, it was found that it contained Fab' with a molecular weight of about 50,000 and protein (complex) with a molecular weight of about 60,000. The anti-1210 IgG fragment is Fab' containing one thiol group, and therefore has one NCS bound to it, and the resulting protein complex is a compound of the present invention where n=1 in the general formula []. It is.

[Brief explanation of drawings]

In FIG. 1, A is a schematic diagram showing the basic structure of immunoglobulin, and B is a schematic diagram showing the structure of IgG1, a human immunoglobulin. FIG. 2 shows the protein efflux pattern of the reaction product obtained in Example 1 (d) in Sephadex G-150 Superfine column chromatography. Figure 3 shows the SDS-
PAGE pattern, disk 1 is anti-
MM46IgG2b antibody, disk 2 is a complex of anti-MM46IgG2b antibody obtained in Example 1 and NCS, and disk 3 is a complex of non-immune IgG2b obtained in Example 1 as well.
This is a band pattern obtained by subjecting the NCS complex to SDS-PAGE. Figure 4 shows Example 1 (E)
These are the results of investigating the antitumor properties of the complex of anti-MM46 antibody and NCS in terms of the therapeutic effect on mice transplanted with MM46 tumors.
FIG. 3 is a graph plotted against the number of days after tumor implantation. a is the case of the complex of anti-MM46 IgG2b and NCS, -●
-: Physiological saline, -○-: 500μg, -△-: 50μg
The figure shows the survival rate when the complex was administered. b shows the survival rate in the case of a complex of non-immune IgG2b and NCS, -●- shows the survival rate when physiological saline was administered, -○- shows 500 μg of the complex, and -Δ- shows the survival rate when 50 μg of the complex was administered. c is anti
In the case of a mixture of MM46IgG2b and NCS at a ratio of 1:1, -●- is physiological saline, -○- is 500 μg, -
Δ- indicates the survival rate when 50 μg of the mixture was administered.

Claims (1)

[Scope of Claims] 1. A selective cell-killing protein complex formed by binding an anti-tumor IgG immunoglobulin or a fragment thereof to neocarzinostatin, represented by the following formula []. [wherein Ab represents an antitumor IgG immunoglobulin or a fragment thereof, and Ab is bonded through an amino group in its chemical structure. NCS stands for neocarzinostatin, and NCS is attached through an amino group in its chemical structure. X represents an m-phenylene group or a trimethylene group, and m represents 0 or 1. S represents a sulfur atom, and when m is 0, S is derived from a thiol group originally contained in the chemical structure of the antitumor IgG immunoglobulin or its fragment;
When m is 1, it originates from a thiol group contained in the introduced organic group. n represents an integer of 1 to 5. ] 2 The following formula [] [wherein Ab represents an antitumor IgG immunoglobulin or a fragment thereof, and Ab is bonded through an amino group in its chemical structure. m represents 0 or 1. S represents a sulfur atom, but when m is 0, S
m is derived from a thiol group originally contained in the chemical structure of the antitumor IgG immunoglobulin or its fragment, and when m is 1, it is derived from a thiol group contained in the introduced organic group. n1 represents an integer from 1 to 5. ] An anti-tumor IgG immunoglobulin having a generated or introduced thiol group represented by or a fragment thereof and the following formula [] [In the formula, NCS represents neocarzinostatin,
NCS is bonded through amino groups in its chemical structure. X represents an m-phenylene group or a trimethylene group] The following formula [] is characterized by reacting neocarzinostatin having an introduced maleimide group represented by the following formula [] [In the formula, n represents an integer of 1 to 5, Ab, m,
S, NCS and X are as defined above.] A method for producing a selective cell-killing protein complex.