JPS58167519A - Cytotoxic complex and preparation thereof - Google Patents

Abstract

NEW MATERIAL:A cytotoxic complex expressed by formulasIand II (Ab is immunoglobulin linkable specifically to a specific antigen or a fragment thereof; B1, B2 and B3 and W are bifunctional organic group; t1 and t2 are 0 or 1; v is an integer 1-10; S1 and S2 are S; Y is amino group or reaction residue of amino group or imino group of a cytotoxic substance containing the amino group or imino group; Z is H or monovalent cation; R1 is H or 1-4C alkyl; R2 is side chain at the alpha-position of an alpha-amino acid, etc.; m is an integer 1-4; n is 5-1500; p and q are 0-1500). USE:An antitumor agent capable of linking specifically to a specific antigen in the target cells, and selectively reacting a cytotoxic substance, e.g. actinomycin D, with tumorous cells. PROCESS:An antibody expressed by formula III or a fragment thereof is reacted with a polymer expressed by formula IV (X is a group capable of forming an active disulfide with the adjacent S) to give a cytotoxic complex expressed by formulaIin which t2 is 1.

Description

[Detailed description of the invention] The present invention relates to a novel cytotoxic conjugate and a method for producing the same.

, more specifically, Fi, a component consisting of an immunoglobulin that specifically binds to a specific antigen of the cells to be killed (hereinafter referred to as target cells), or a fragment containing its antigen-binding site, and a cytotoxic substance. The present invention relates to a novel cytotoxic complex having a component made of a polymer and a method for producing the same.

□1 Conventionally, it is known that an antitumor agent can be produced by binding an antitumor antibody to VCm cytotoxicity.

For example, in JP-A No. 58SL-61840 KFi, an antitumor immunoglobulin is reacted with an anticancer drug having an amide 7 group or a carboxyl group in the molecule in the presence of water-soluble carbodiimide, and 5 to 15 molecules per immunoglobulin molecule are reacted. It is disclosed that an antitumor agent has been obtained in which an anticancer agent is bound with an amide bond. In addition, JP-A-S 1-144723 describes anti-tumor immunoglobulin IPab as an anti-tumor antibody.
Using a 'z-mer, an anticancer drug (e.g., daunomycin) oxidized with sodium monopergorate is bound to the free amino group of this to form a Schiff base, and the resulting bond is then stabilized with sodium borohydride. Accordingly, F
It is disclosed that an antitumor agent consisting of an ab' dimer and an anticancer agent was obtained. Furthermore, JP-A-1@S 1-12628
No. 1 contains a polymer ff in which antitumor immunoglobulin and 5 to s"o"o molecules of an anticancer drug are covalently bonded per molecule.
It has been disclosed that antitumor agents were obtained by linking i-forms (eg, poly'glutamic acid) through amide bonds.

Antitumor agents obtained by these methods are expected to bind selectively to tumor cells and exert toxicity to tumor cells, making them extremely interesting drugs. When binding to antibodies, immunoglobulin 17
When a large number of anticancer drug molecules are bound to a tumor, the antigen recognition activity of the antibody decreases, so in order to avoid this difficulty, it is necessary to limit the number of anticancer drug molecules to a small number. However, in the latter case, the company faces the difficulty that the produced antibody-drug conjugate does not have sufficient cancer cell-killing ability.

On the other hand, when a polymer is used as a drug carrier, it is thought that the above-mentioned difficulties can be improved. but,
Note for JP-A No. 51-126281 - Because the reaction of binding a large number of anticancer drugs to a polymer carrier and the reaction of binding an antibody to a polymer-anticancer drug conjugate are the same reaction,
Problems arise because a large number of antibody molecules bind to the polymer carrier, and the resulting complex not only cannot be homogeneous, but also contains high molecular weight substances that are unsuitable for use as therapeutic agents. be.

As a result of intensive research to solve the drawbacks of the prior art, the present inventors have discovered a method for binding drugs that contains only 1 reactive group for binding reaction with antibodies and is different from that. , prepare a polymer carrier containing a large number of reactive groups,
By first binding a large number of drugs to the polymer carrier using a large number of reactive groups, and then bonding them to an antibody using a reactive group with the antibody, a high The inventors have discovered that it is possible to produce an antibody-drug complex that does not contain molecular substances and binds a large number of drugs, and has achieved an uninvention.

That is, the present invention provides the formula [1] in which an immunoglobulin or its 7 fragments capable of specifically binding to a specific antigen possessed by cells to be killed is covalently bonded to a reactive polymer bound to a cytotoxic substance. ... Mortar] #l1lll toxic complex represented by and formula [11]
...[1] Cytotoxic complexes represented by the following, as well as their production methods VC@.

In the present invention, the immunoglobulin (inducer of cytotoxic complex) that can specifically bind to a specific antigen possessed by cells to be killed, represented by Ab in formula [1], is the following book. It is.

Humans immunized with tumor +111 cells or target cells such as specific lymphocytes or tissues containing them.

Ethanol fraction from antisera isolated from animals such as monkeys, horses, cows, goats, sheep, rabbits, guinea pigs, hamsters, runts, and mice.

Immunoglobulin produced by known means such as ammonium sulfate fractionation, ion exchange, or molecular sieve column coomadography, or antibody-producing cells collected from animals immunized with target cells are made cancerous with carcinogenic substances. A monoclonal antibody produced by a hybridoma obtained by fusion with a myeloma cell or a myeloma cell. Further, immunoglobulins specific to target cells, which are obtained by separating immunoglobulins bound to target cells using a surfactant or the like, are also included in the uninvented immunoglobulins.

Immunoglobulins include IgG, gA, IBM, and Ig.
D.

Five classes of IgIC are known, and each class is further known to consist of several subclasses. However, their basic structures are the same in that they consist of two heavy chains and two light chains, as well as a Fab portion with antigen-binding activity and a Pc portion with effector activity. However, 1gM is s-mer, IgA
Some of them exist in the form of dimers, but considering the tissue permeability of the Ia syphilitic complex, these can be reduced with mercapta/! It is preferable to use it as a polymer and then use it as the guiding part of the complex.

Although the entire immunoglobulin molecule may be used as the inducing portion of the cytotoxic complex, it is preferable to use a fragment that contains the antigen-binding site but does not have the Fc portion. In a complex containing an Fc moiety, non-specific adsorption to cells other than the target cell by the Fc moiety and binding to the Pc'J receptor on the cell membrane occur, and the cytotoxic complex selects cells to be killed. This is because the sex is diminished. Furthermore, since the antigenicity of immunoglobulin as a heterologous protein is strong in %IC in the Pc moiety, the 7 fragments of immunoglobulin without the IFo moiety are effective in inducing the m-syphilitic complex in terms of reducing the antigenicity of the complex. In general, immunoglobulins are preferably combined with papain,
Trypsin.

When it is degraded with a proteolytic enzyme such as chymotrypsin or plasmin, a so-called Fab 7 fragment having one antigen-binding portion is obtained. Also, pepsin degradation.

Depending on the conditions, a so-called y(ab')H fragment having two antigen-binding portions can be obtained by trypsin digestion. This fragment is further treated with a mercaptan to become a -valent Fab' fragment. Furthermore, when immunoglobulin is denatured and degraded, antigen-binding regions (variable regions) are formed.
egion ) is obtained.

These immunoglobulin-derived fragments are derived from any class of immunoglobulin used as a raw material.

Any subclass can be used as the derivative of the complex of the present invention.

In the cytotoxic complex represented by formula [1], Y represents an amino group- or imino-reactive residue of a cytotoxic substance containing an amino group or imino group in the molecule. In the present invention, a cytotoxic substance refers to a substance that exhibits toxicity to cells in its original state, or a substance that does not exhibit toxicity as it is, but can be converted into a substance capable of exerting toxicity to cells in vivo. Examples of these include p-[N,N-bis(2-chloroethyl)]phenylenediamine p-' [bis(2-chloroethyl)amino]L-phenylalanine (melphalan) 2-agino'M-
[p-bis(2-chloroethyl)aξno]phenyl3
-Hydroxyglopionamide i-sen) H 1-(β-D-arabino75novir)cytosine t or its monophosphate 1-[5'-(2-aminoethylphosphoryl)-β-D
-arabinofuranosyl]cytosine C 01 (2-Amino-N-[p-bis(2-chloroethyl)aminocophenyl-3-hydroxy-2-hydroxymethylglopionamide 00H Methotrexate actinomycin D 011 Mitomycin C Examples include Sir H, Daunomycin X Catfish OH, and Adriamycin.

2 is a hydrogen atom or a monovalent cation, such as Ha+,
+, NHa+.

W represents a divalent organic group and is not particularly limited as long as it is an inert group that does not participate in any reaction during the process of obtaining the reactive polymer used as the raw material of the present invention. These groups include, for example, 2-aminoethanethiol residue (-CkhC
Examples include straight-chain phenylene groups such as Hs-), cysteine benzyl ester residue alkylene groups, and phenylene groups with or without substituents such as 4-aminothiophenol residues (Q); Particularly preferred are 1 to 4 alkylene groups.

R+ is a hydrogen atom or an alkyl group having 5 to 4 carbon atoms, preferably a hydrogen atom.

R is the 6-position side chain of an α-amino acid or a derivative thereof (excluding groups with a carboxyl group, for example,
If the amino acid is glycine, it is 9wm ii, if it is alanine, it is Rt-OFIm, if it is phenylalanine, it is Rt-OHtD, if it is serine, it is Rt-O&OH.
It is. In the complex of formula [1], the unit consisting of the a-amino acid does not participate in any way in binding with the cytotoxic substance, but it improves the fat solubility and water solubility of the complex, the iK compatibility with the S membrane, etc. It is useful for adjusting. Therefore, in cases where there is no particular need to adjust fat solubility or water solubility, a method that does not contain such a-amino acid units (formula [1)]
q-0) is practically advantageous.

m represents an integer of 1 to 4, preferably m is 1 or 2. In addition, the complex represented by 2m includes, for example, a polymer in which m-1 and mm2 are mixed. where p represents the number of structural units to which no cytotoxic substance is bound, and q represents the number of α-amino acid units (the side chains may be modified) that do not have a carboxyl group on the side chain, The arrangement state in these basic polymers is arbitrary.

That is, in formula [1], for convenience, it is expressed as a block polymer, but it is not limited to this, and polymers obtained by ordinary methods are randomly arranged polymers. n-s ~ 150G, preferably tG ~ 500,
pwQ~1500, preferably oNsoo, q-
o~150G.

Preferably it is q-0-SOO.

8I and Bt represent sulfur atoms, which are derived from the thiol group or active disulfide group generated or introduced into the antibody represented by 8+aAb or its 7 fragment, and fh is the reactive group bound to the cytotoxic substance. B1 and Bw, which represent sulfur atoms derived from thiol groups or active disulfide groups on the polymer, represent only two organic groups, which can be used to form a reactive polymer with a cytotoxic substance bound to an antibody or its 72 segment. This book is derived from the crosslinking agent described below that can be used when bonding.

When the cytotoxic complex represented by formula [I] in the present invention is smQ, that is, when the cytotoxic complex is represented by the following formula (1-1). The complex has the following formula [厘] Al:+(-(B→＠,
8 tFL ] v
・・・・・・ Antibody expressed by [厘] or its 72
and a reactive polymer having an active disulfide group represented by IIQ and bound to a cytotoxic substance, can be produced by the following formula CW. ff) In,
X represents a group capable of forming an active disulfide bond with a hydrogen atom or a sulfur atom;
), 2-benzothiazoylthio group (α>8-)
+ 2-Peto-phenylamino-N'-phenyliminomethyl formula (1
The cell-mediated complex represented by -11 also contains an antibody or its fragment represented by the following formula [v] (same as combination), which has an induced or introduced active disulfide group, and the following formula [W ] co aooz (W) It can also be produced by reacting a reactive polymer having a thiol group and bound to a cytotoxic substance.

In the present invention, in the cytotoxic 1 complex represented by the formula [11], when t, -1, that is, the cytotoxic complex is represented by the following formula [1-*] ... [1-2] In this case, an antibody represented by the formula [厘] or its 7 fragments having a generated or introduced thiol group, and a reactive polymer having a thiol group to which a cytotoxic substance represented by the formula [■] is bonded. This can be produced by bonding two functional groups capable of reacting with thiol groups using a crosslinking agent. As a crosslinking agent that can be suitably used for this purpose, [B4 is 2
It is a valent organic group. ] Crosslinking agents or benzoquinone can be mentioned. Specific examples of the crosslinking agent represented by the formula [■'] include N,)f'-(1,2-phenylene)4.4'-
Bis(maleoyl amino) azope/zen can be mentioned.

When this reaction is carried out, it is preferably carried out as a two-step reaction. That is, first, an excess of, for example, a reactive polymer having bound to an antibody represented by the formula [a+] or its 7 fragments, or a cytotoxic substance represented by the formula [W], A reaction intermediate having a limide group represented by formula 1 [■] or formula [■] co cooz 7 . . . [■] 2 is obtained by performing a purification treatment. Next, for the reaction intermediate [■] K, a reactive polymer bound with a cytotoxic substance represented by the formula [1] was added, and for the reaction intermediate [K] The expressed antibody or fragment thereof is allowed to act. By the above reaction procedure, a substance corresponding to the formula [1-4] K can be produced using one target substance.

In the present invention, in order to produce the JII cytotoxic syphilis complex bound to a cytotoxic substance and represented by the following formula:
This can be achieved by reacting an antibody having an introduced maleimide group and represented by the following formula [■] or its 7 fragments with a reactive polymer having a cytotoxic substance bound thereto and represented by the formula [W]. can.

The reactive polymer bound to the cytotoxic substance represented by the formula (W) and the formula [1], which is the raw material for the production of the cytotoxic complex represented by the formula [1] and the formula [Nihon], was developed by the present inventors. Invention by (Japanese Patent Application Laid-Open No. 57-9724 and 57-18727)
It is manufactured by method 3, and the outline thereof is as follows.・.

For example, glutamic acid benzyl ester (r-penzyl-L-glutamic acid) is reacted with phosgene to obtain r-benzyl-L-glutamate N-carmenic acid anhydride, which is then converted into, for example, cystamine (khMOF1*0F1tB8
When a polymer is obtained by polymerization using Qyama cutaa snow) and this polymer is subjected to acid decomposition or alkali decomposition, a polymer of the following formula is obtained.

9&CO鵞H Next, this product is treated with mercaptan to cleave the disulfide bonds, and the following poly L-'glutamic acid having a thiol group at the terminal is obtained from K.

CH Do00*H Next, is this KI? ! By using an i-type disulfide compound, it is possible to obtain the following poly-L-glutamic acid having an active disulfide group at the terminal.

When such terminally activated poly-L-glutamic acid is reacted with a cytotoxic substance containing an amino group in the presence of a carbodiimide reagent, the following formula 0 (formula It is possible to obtain a reactive polymer having an active disulfide group and bound to a cytotoxic substance, as shown in (Y in the middle represents a cytotoxic substance residue). Furthermore, if a thiol group is generated by treating this material with a mercaptan, a reactive polymer bound to a cytotoxic substance having a thiol group, as shown by the following formula 0vki corresponding to the formula [1] K, can be obtained. can be obtained. Therefore, a reactive polymer bound to a cytotoxic substance represented by formula [1] can be obtained by allowing a mercaptan or a borohydride compound to act on a reactive polymer bound to a cytotoxic substance represented by formula [W]. Next, we will describe a method for generating or introducing a thiol group into antibodies (immunoglobulins) or fragments thereof, which can easily be obtained by reductively cleaving disulfide bonds.

First, a method for generating a thiol group in immunoglobulin or its 7 fragments, that is, in the formula t,
A method for producing an antibody corresponding to -o1c or its 7 fragments will be described. Immunoglobulins include egG, egA,
Engineering gM.

Five classes are known: IgD, Igl, IgM, iiS-mer, ll(A), and some z-mers.

Dithiol groups can be generated by reducing these with mercaptan to form l-mers. In addition, for example, when a certain type of saccharide is applied to G-G by the above-mentioned method, that is, trypsin decomposition and mercaptan treatment, Fab' having one thiol group can be obtained from K.

Next, a method for introducing a thiol group into an immunoglobulin or a fragment thereof, that is, in formula [11], t, -
The antibody corresponding to IK or its 7 ragmen) f) Production method will be described. This can be accomplished by reacting the immunoglobulin or its 7 fragments with a suitable cross-linking agent and, if necessary, subjecting it to further suitable treatment. Examples of crosslinking agents that can be suitably used for this purpose include the following formula [Xl Xl-8-81-BS-(!-Q+...
・[X] A crosslinking agent represented by 1, the following formula [Summer] 1m-8-lm-8-8-B... (:) [)1 A crosslinking agent represented by 1-HL, the following formula [Expletive] H8-BS -0-Qt...
[Expletive] A cross-linking agent represented by the following formula [Xll], a cross-linking agent (N-acetylhomocystine) represented by the following formula [Xll],
Examples include a crosslinking agent (S-acetylmercaptosuccinic anhydride) represented by the following formula [mushroom]. When treated with these crosslinking agents, a thiol group can be immediately introduced into the immunoglobulin or fragment thereof, i.e., the formulas [Xff), [■],
In cases other than when using a crosslinking agent represented by [π],
In other words, when processing with the expression ['X], [summer], [with],
Furthermore, when the immunoglobulin or its 7 fragments are treated with a mercaptan or an alkali, respectively, a thiol group can be introduced onto the immunoglobulin or its fragment.

Next, a method for inducing or introducing a k-active disulfide group in an antibody or a fragment thereof will be described. First, a method for generating K-active disulfide groups in immunoglobulins or fragments thereof.

That is, a method for producing an antibody corresponding to t, -OK in formula [v] and its 7 fragments will be described.

The above-mentioned parts of IBM and GM or the yat/7 fragment generate thiol groups, and this can be achieved by reacting these with the active disulfide compound described below.

Next, a method for introducing active disulfide groups into antibodies or fragments thereof will be described. An active disulfide group-introducing agent represented by formula (X), formula (Xl, etc.) (antibody or fragment thereof) is acted on, or
], [XI], and [lff] to introduce a thiol group into the antibody or its 7 fragments, and then react with active disulfide compounds such as these Kvk. can.

Examples of active disulfide compounds that can be used for the above purpose include 2-pyridyl s, disulfide Y
(c) P-s-808), 5.5'-cythiobis(
2-Nitrobenzoic acid) N-oxy-2-pyridyl sulfide ↓ ↓ 0 2-Benzothiazoyl disulfide N-phenylamino-N'-phenyliminomethyl can be substituted.

By sulfonating the disulfide bond of the hinge portion of P (at/)t of Moejo, for example, with sulfite ion, yet/ having a KB-sulfo group (-s-sor) in the molecule can be obtained. But this thing too.

A cytotoxic complex represented by formula (1-11) is produced by suitably reacting with a reactive polymer bound to a cytotoxic substance represented by formula [■].

Next, a method will be described in which a maleimide group is introduced into an antibody or its 7 fragments to obtain a reaction raw material represented by the formula [■].

This can be accomplished by reacting the antibody or its 7 fragments with a crosslinking agent represented by the following formula [bi] 0 0 and performing operations such as purification. Specific examples of crosslinking agents represented by [store] are as follows.

Production of a cytotoxic complex in the present invention by reacting the modified antibody (immunoglobulin) and its 7 fragments with a reactive polymer bound to a cytotoxic substance is carried out under the following conditions.

The reaction is performed at the PH5 of the modified antibody and its fragments.
-9 concentration in the buffer solution is O, S ~ 100q/- (more preferably 1 ~ zosp/d) K [I14 prepared solution and an aqueous solution of a reactive polymer bound to a cytotoxic substance, Alternatively, if the same buffer solutions mentioned above are mixed and left as is or stirred gently, the reaction proceeds from K.

As for the mixing amount, it is preferable to use the polymer in an amount of o, t - to molar amount based on the molar number of the antibody. Reaction temperature is 0-5
It is 0°C. Reaction time is reaction scale.

Although it depends on the reaction conditions, it is generally within 2 days.

Separation and purification of the complex from the reaction mixture can be carried out using commonly used operations 1, such as molecular sieve column chromatography, dialysis, and Affinity chromatography.

In the reaction for producing a cytotoxic complex consisting of a polymer in which an antibody or its 7 fragments are bound to a cytotoxic substance, when reacting the antibody or its fragment with a crosslinking agent, for 1 mole of protein to be reacted with the crosslinking agent, It is preferred to use 1 to 100 moles of crosslinking agent. The reaction involves the addition of antibodies or fragments thereof to protein ammonium chloride in a buffer at pH 5-9.
is 0.5 to 0.5 l OOK9/Ill (more preferably 5 to 20 ■/d) K. Add aqueous solution of the crosslinking agent or the crosslinking agent does not dissolve in water while stirring at 0 to 50°C. In this case, the crosslinking agent is mixed with a small amount of an organic solvent such as N,N-dimethylformamide, dimethylsulfoxide, l,2-dimethoxyethane, methanol,
This is done by adding a solution dissolved in ethanol, acetone, etc.

The reaction time depends on the scale of the reaction and the reaction conditions, but is generally within 2 days. The modified antibody is purified by removing low molecules by dialysis or molecular sieve column chromatography.

A brewing complex represented by the formula [W] containing a thiol group,
For example, when reacting a crosslinking agent represented by formula [■'], this can be carried out under similar conditions.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example! (b) Preparation of mouse leukemia Lt210iC-specific immunoglobulin 1 mouse leukemia cell L passaged in DBA/2cr mice
1210 was extracted from the ascites of a DBA/20Fw mouse, and about 10 pieces thereof were made into an emulsion with 70 India complete adjuvant and intravenously injected into domestic rabbits. after that! K, 3 times at 1-week intervals, approximately 10 pieces each (QL
12101@ cells were injected subcutaneously with Aziyuvanod, and blood was collected 7 and 10 days after the final dose H. The obtained blood was pooled, the serum was separated, and the serum was incubated at 56°C for 3
It was heated for 0 minutes to immobilize it.

To 20 (1 m) of the anti-L1210 serum thus obtained, 200 m of a saturated aqueous solution of ammonium sulfate was added, and the resulting precipitate was separated by centrifugation. dK folding, fK folding II
Dialyzed thoroughly against the solution. This dialysate was equilibrated with the same buffer using DIeAE cellulose column chromatography (column size 3 esm x 94 exa
) K multiplied by the unadsorbed amount - anti-L1210 IgG
A solution containing was obtained.

(b) Separation of F(at1') fragment from immunoglobulin 1. of anti-L12101gG obtained as in 0) above.
21F in 0.1 M acetate buffer (pH 4,5) 40i
After digestion at 37 °C for about 18 hours by adding 24 inches of pepsin, the degradation products were subjected to Sephadex G200 column chromatography in physiological saline (column size 3.5 am, × 140 cm). K was applied, and the protein flowing out at a molecular weight of approximately 100,000 was extracted. When this is multiplied by 8D8-PAGICK, it becomes pure F(a
b')* fragment was confirmed.

(c) Preparation of Fab' fragment 0.01 M Tris-HCl containing 26 inches of P (ab')s fragment obtained as above (→),
14M sodium chloride-2WE Mml:DTA solution (
pH (8,3) 1. 0.2 d of 15mM 2-mercaptoethanol aqueous solution was added to the oil, and the mixture was reduced at 37°C for 1 hour. After the reaction, the bath solution was diluted with 5WLM acetate buffer.
0,14M sodium chloride-1m MKDTA solution (p
Sephadex G 25 (Q
, 8 CmX 4'Ocm) to remove z-mercaptoethanol, to obtain a Fab' fragment having a thiol group tat.

) Fab' fragment (1) and active disulfide.

Preparation of the cytotoxic conjugate (3) by reaction with poly-L-glutamate (2) containing a daunomycin group and conjugated with daunomycin.

Mountain C Foil 1 To C00 Na ■ Y+ (2) C mountain C mountain 1 C mountain C mountain 1 Co Co Na (3) Solution of Fab' fragment obtained as in (c) above (11,1897M) l-K, solution of the sodium salt of poly-seaglutamate (2) having active disulfide groups and conjugated with daunomycin (active disulfide equivalent t, 1 x 1 o-'mole /d) 2.2-
and 0.1 M sodium chloride 2111MII
5011M glycine sodium salt buffer (pH 9,2) K containing iDTA.

The reaction was carried out at 4° C. while being dialyzed for 48 hours to obtain the desired complex. The reaction product was further dialyzed against 11 (10,1 il-sodium chloride solution).

a of the complex (see) for GtIL t 21 G cells
target cell L of complex (3) obtained as above)
The cytotoxicity against 1210 was investigated.

24 large culture plays) 1+1:, 5X10 L1
Dispense 0.9 d of RPMI 1640 medium (containing 100% tallow serum, 20 μM 2-mercapto-ethanol and O,IQ/- kanamycin) containing 21G cells, and add test sample 0 at the desired concentration. After 48 hours of culture at 37° C. in a Cot atmosphere, the number of living cells was measured by Torinosin blue staining.

As a result, as shown in Table 1, the complex showed a concentration-dependent cell proliferation inhibitory effect on the target cell L+1210. The culture was carried out in two series, and the values are shown as the average.

Example 2 (a) Preparation example t of XgG introduced with active thiol group
0.00 containing 1gG2.4 staves obtained as in oeo.
1M phosphate buffer (contains 0.1M sodium chloride,
Ethanol O,OS* containing 1.6 WLM of N-succinimidyl-3-(2-pyridylthio)propionate was added to pH 7.5) 2-, and the mixture was reacted at room temperature for 30 minutes with occasional stirring. Next, a column of Sephadex G-25 equilibrated with the above buffer solution (o, 8℃ m x 4g
The reaction solution was poured onto C.C.) to remove excess reagent. In this way, 1gG containing 91.8 3-(2-pyridyl)dithiopropionyl groups per molecule was obtained.

(b) Production of a cytotoxic complex (see) by the reaction of dithiopropionyl 1gG (4) and poly-seaglutamate (5) which has a thiol group and is bound to daunomycin. ) Poly-seaglutamate (5 ) solution (terminal BH equivalent 1.I X lo-'mole/d) G, 05
The complex obtained in this way (6
) was concentrated (by ultrafiltration) and dialyzed against 0° sl sodium chloride solution.

(Cytotoxicity of the complex against 9L l 210 Jl cells) The target g of the complex (6) obtained as described above
The syphilitic effect on L l 2 i 0 K was investigated according to the method described in Example 1 above.

As a result, as shown in Table 2, 1
Upon addition of complex (6) of sM.

The proliferation of L121G cells was suppressed.

Table 2 Example 3 (a) Preparation of F'(ab) snow into which a maleimide group has been introduced
1011M phosphate buffer (PH6,5) containing ■1.1
4 sdK, M-hydroxysuccinimidyl-m-
0.05-M,M-dimethylformamide containing 0.42 wg of maleimidobenzoic acid was added and reacted at room temperature for 40 minutes. After the reaction, 0.14 M sodium chloride, II
IM-51 °C M acetic acid mild solution containing original DTA (pHs, s
) Excess reagent was removed by Ic dialysis to obtain m-maleimidobenzoyl F (ab')@.

←) H-w leimidobenzoyl'F(ab')l('L
) and a poly(L-glutamer)-L-alanine copolymer (8) with thiol groups and melphalan attached to form a cytotoxic complex (9) to COC, Na 1” to C. 7) A solution containing 6.9 mg of 9m-maleimidobenzoyl F(ab') obtained as in 0) above, 7s
dK, an acetate buffer containing a poly L-glutamate-L-alanine copolymer (8) having a thiol group and bonding melphalan (the above pi containing the terminal SR phase 8.OXl 0-'mole /d) Add 0.47a/ of liquid and further add 0.47a/
．． 0.15 d of 6MIJ acid buffer (pH 6.5) was added, and the mixture was allowed to react at 4°C for 16 hours. The complex (9) thus obtained was dialyzed against 0.91+ sodium chloride solution.

Cytotoxicity of the complex to HL s 21 G cells Target cell L1 of the complex (9) obtained as in (b) above
Cytotoxicity against 21OK was investigated.

In centrifuge tube: R containing 5X104 L12'IO cells.
PM164G medium (to% tallow serum.

Dispense 0.9- (containing 20*M 2-mercagtoethanol and 0.1η/- kanamycin), and then add 0.1- of the test sample at various concentrations =j for 20 minutes at 37°C. After pre-incubation, centrifuge, remove the supernatant, add the above medium 1-, and add 1! The cells were cultured for 48 hours at 37°C under an OCh atmosphere. After culturing, the number of viable cells was measured by trypan blue staining.

As a result, number 3! ! As shown in melphalan phase 4io
When incubated with the complex of oμ, Ll
, 210 cells showed an inhibitory effect on proliferation. Since the effect was seen even though the reagent was removed after 20 minutes of incubation, it is thought that a considerable degree of binding between the complex and the cells occurred during the initial 20 minutes. The culture was carried out in two series, and the values are shown as the average.

Example 4 (a) Preparation of Fab' with maleimide group introduced Free thiol group 1 obtained as in (c) of Example 1 above
5111M acetate buffer (0.14M sodium chloride.

1-1111-1l1, pH 5,5) 5.7
'd and 1.219 of 0-phenylene dimaleide.
After mixing with rM buffer 2- and reacting at 30°C for 60 minutes, the mixture was dialyzed against the same buffer to remove excess reagent.

(b) A cytotoxic complex (1
2) Manufacturing.

(10) To COC Na Y4 (1) 0-phenylene simaleisyl F obtained as above
7. The buffer solution of 0) above containing azo(10) 9.8 Q.
Poly-seaglutamate (11) with a thiol group and ara-C bonded to 8- (terminal BH equivalent a, ox
The same buffer containing t o-'mole/d) 0.67
1d and 0.5M phosphate buffer (pH 6,5) o, aw
J was added and reacted at 30°C for 60 minutes.

The complex (1z) thus obtained was further dialyzed into a solution of K2O,99)IJ.

→ Cytotoxicity of the complex against L121G cells (above)
Target cell L1 of complex (12) obtained as in b)
Cytotoxicity against 210iC was investigated according to the method shown in Example 3 f→ above.

As a result, as shown in Table 4, when pre-incubation was performed with 10 jM of a complex equivalent to AraO, it showed moderate cytotoxicity to L1210 cells.

Claims (1)

[Scope of Claims] 1. A cytotoxic complex represented by the above formula [11].・・・・・・〔! ] 2. A cytotoxic complex represented by the following formula [1] % formula % [111]. 3. 2m and the formula [fll, W has 1 to 1 carbon atoms
4 is an alkylene group. A cytotoxic complex according to claims 1 and 2. 4. The following formula [[
] An antibody represented by the formula % or its 7 fragments and the following formula ['
M] 0) 0OOZ] Y... [W] Characterized by reacting a reactive polymer bound with a cytotoxic substance represented by the following formula [1-1] % formula %] A method for producing a cytotoxic complex. 5, #The following formula (Vl Ab ++B+)H8+X]v having an introduced or introduced active disulfide group
-...=Antibody represented by [V] or its 7 fragments and the following formula 1j! ] -...[%] Reacting with a reactive polymer bound to a cytotoxic substance having a thiol group, expressed by the above formula t:t-t] A method for producing a cytotoxic complex. 6. The above formula having a generated or introduced thiol group [
The antibody represented by [II] or its 7 fragments, and the reactive polymer having a cytotoxic substance having a thiol group, which is represented by the above formula [], and which have two functional groups capable of reacting with a 1,000-ol group. A method for producing a cytotoxic complex represented by the following formula (1-23% formula % [12]) characterized by bonding using a crosslinking agent. (2) A cell represented by the formula [l], which is characterized by reacting an antibody represented by the formula [1] or its 7 fragments with a reactive polymer bound to a cytotoxic substance having a thiol group, represented by the formula [V]. Method for producing toxic complexes.