JPS6119635A - Cytotoxic substance and protein complex - Google Patents

Abstract

PURPOSE:The titled complex useful as a target-directing carcino-static agent, prepared by binding a physiologically active protein with a specified polysuccinimide derivative through mercapto groups. CONSTITUTION:A cytotoxic substance/physiologically active protein complex is obtained by binding the mercapto groups of a physiologically active protein (e.g., urokinase) with the mercapto groups of a polysuccinimide derivative of an average MW of 2,000-1,000,000 comprising the structural units of formulas I -VI [wherein X is a reaction residue of a cytotoxic substance having an amino or imino group in the molecule, Y is a water-soluble aliphatic primary amine residue, Z is H or a group of the formula: Z' (when Z is H, one -SH group represented by -SZ, together with another -SH group can form an intramolecular or intermolecular -S-S- bond in equilibrium, these are represented inclusively by -SH), and Z' is a group which, together with an adjacent S, can form a reactive disulfide], wherein the numbers of said structural units of formulas I -VI are m, l, r, p, and q which are each 0 or a natural number and satisfy the relationships of formulas VII and VIII.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to complexes of cytotoxic substances and physiologically active proteins and methods for their production. More specifically, the present invention is directed to a combination of a cytotoxic substance and a physiologically active protein that is useful as a target-directed anticancer agent by binding a cytotoxic substance to a physiologically active protein such as an antitumor antibody that has the ability to bind to a target substance such as a tumor cell. This invention relates to a composite and its manufacturing method.

It has been known to produce an antitumor agent by binding an antitumor antibody to a cytotoxic substance. Polymer carriers are used to bind a large number of cytotoxic substances without reducing the target binding ability of the antitumor antibody.

Typical polymers used as polymer carriers are dextran and polyglutamic acid.

In the case of dextran, it is mainly used as an aldehyde derivative by oxidation with periodic acid, etc., but it is difficult to obtain a conjugate with good reproducibility because the cytotoxic substance and antitumor antibody are reacted with the same functional group. Anti-tumor antibodies bind more than necessary and produce high molecular weight substances, which tends to reduce the ability to bind to tumor cells and the like. Moreover, polyglutamic acid has the above-mentioned drawbacks and has other charges, which lowers the binding ability of antibodies and at the same time collects in specific organs in the body, making it undesirable as a target-directed anticancer agent.

The present inventors conducted intensive studies to solve these drawbacks, and as a result, they synthesized an uncharged polymer carrier to which a cytotoxic substance was bound. It was discovered that a complex of a cytotoxic substance and a bioactive protein such as an antitumor antibody can be obtained by binding to the cytotoxic substance, leading to the present invention.

The present invention provides a complex between a reactive polymer bound to a cytotoxic substance and a bioactive protein such as an antitumor antibody having targeting ability, which can be used to produce a target-directed therapeutic agent.

That is, the present invention provides the following general formula [
1] to [6] (3) (4) [In the above formulas [1] to [6], x, y, w and 2
have the following meanings.

X: Reactive residue of a cytotoxic substance having an amino group or imino group in the molecule Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group 2: Hydrogen atom or group represented by the general formula Z' (however, ,2
is a hydrogen atom, the -SH group represented by -SZ is in equilibrium with other 15H groups within or between molecules.
Although bonds may be formed, these are included in the expression -8H. ) Z′: a group capable of forming an active disulfide bond with the adjacent sulfur atom] and each of the structural units in one molecule (1), (2], (3)', (
4) The numbers of (5) and [6] are n and m, respectively.
, l! , r, p and 9, these numbers are Ol or natural numbers, and the relationship between them is n-4-m≧2 0.001≦(p+q)/(n+m+r+r+p+q)
= o, s.

and the average molecular weight is 2,000~i, ooo, oo
This is a protein-vorosuccinimide complex in which the polysuccinimide derivative (1), which is o, is bound to a physiologically active protein.

Here, as the chain group (2') that forms an active disulfide bond with the adjacent sulfur atom, for example, a dilthio group (il
)"s-), a substituted or unsubstituted pyridylthio group such as 5-carboxy- or its N-oxide, and 3-
Carboxy-4-nitrophenylthio groups, and N-phenylamino-N'-phenylimino W are lower alkylene groups, such as linear or branched alkylene groups having 1 to 4 carbon atoms. can be mentioned.

In formulas [1] and [2], X represents a reactive residue of a cytotoxic substance containing an amino group or an imino group in the molecule or a hydroxyl group.

A cytotoxic substance in the present invention refers to a substance that exhibits toxicity to cells as it is, or a substance that does not exhibit toxicity as it is but can be converted into a substance capable of exhibiting toxicity to cells in vivo. Examples of these include:

p-(N, N-bis(2-chloroethyl)]phenylenediamine p-(bis(2-chloroethyl)amino)-L-phenylalanine (mel) 1 run) 2-amino-N-(p-bis(2- chloroethyl)aminocophenyl-3-hydroxypropionamide 1-(5'-(2-aminoethylphosphoryl)-β-D
-arabinofuranosyl] long tocytosine 2mino-N -[
p-bis(2-chloroethyl)amino]phenyl-3-
Hydroxy-2-hydroxymethylpropionate tF Methotrexate actinomycin D Mitomycin C X and H, daunomycin . (J+r-0) is not necessarily used for the purpose of the present invention, but is introduced for the purpose of improving the water solubility of the reactive polymer (polysuccinimide derivative [I]). Examples of water-soluble aliphatic primary amines used include ethanolamine, aliphatic primary amines having a hydroxyl group such as 3-amino-1,2-propanediol, (J(3
(CH2)gM (2 (an integer of g-i to 4)) aliphatic amine, etc. Especially preferred are amines having a hydroxyl group such as ethanol, amine, and 3-amino-1,.2-propanediol.

It represents the number of acid units, and p+q represents the number of constituent units to which 2 is bonded.

(n+m)/(n+m-1+r+p+q) represents the number of condensed moles of X per mole of succinimide group, which is a monomer unit.

<p+q>/<n+m+t+r+p+q) is the general formula for 1 mole of succinimide group, which is a monomer unit.
-Nu-WS-Z represents the number of condensed moles of the group.

The molecular weight was expressed as the value obtained by quantifying the terminal amino group.

The arrangement of the structural units in the polymer is arbitrary. Examples include random copolymers, but are not limited thereto.

Molecular weight is 2,000 to a, ooo, ooo preferably 2
．． 000 to 3,000,000, n0m is 2 or more, (n+m)/(n+rn+l+r+p+q) is less than 1: (p+q)/(n0m+/10r+p+q) is 0°001 or more, 0. It is 50 or less, preferably 0.3 or less.

If the molecular weight is less than this, the number of cytotoxic substances bound to one molecule of the reactive polymer decreases, and the amount of cytotoxic substances that can bind to anti-tumor antibodies etc. decreases, which is not preferable. Furthermore, if the molecular weight is too large, the anti-tumor antibody aggregate becomes less viable, making it difficult to produce insolubilized components and large molecular weight, which is not preferable.

(p1q)/(n+m+/+r+p+q) is 0.001
If it is less than this, the binding ratio with anti-tumor antibodies etc. will be low, which is not preferable. Further, it is less than 0°5, preferably less than O63, and if it is too large, the aggregates such as anti-tumor antibodies become less viable and the insolubilized content and large molecular weight content increase, which is not preferable.

The method for producing the polysuccinimide derivative [I] in the present invention is shown in Japanese Patent Application No. 59-25665.

That is, polysuccinimide (polysuccinimide 1:]I) whose main component is a structural unit of the following general formula (II)
can be more easily induced.

[In the formula, k represents a natural number. ] Polysuccinimide is a polymer also known as anhydropyasparatic acid, and can be obtained mainly by thermal condensation of aspartic acid. (
J., Kovacs et al., J.

Org, Chem, 261084 (1961);
P., Neri et al.

J. Med. Chem. 16 893 (1973))
As an example of a specific production method, aspartic acid (which may be an optically active form or a DL form) is mixed with 16 ml of aspartic acid under normal pressure or reduced pressure.
It can be obtained by dehydration polymerization at 0'C or more and 240C or less.

After polymerization, the reaction product is dissolved in DMF or DMSO# and reprecipitated in water to remove unreacted aspartic acid and low-molecular condensate, filtered through a glass filter, and then dried.

Repeat this operation if necessary.

Although IR and NMR suggest that the polymer thus obtained is represented by the general formula [II], it is substantially the same as the general formula (II) even if it partially contains other constituent units. It is generally accepted that the structural unit of [II] is the main component.

There are two methods for obtaining the polysuccinimide derivative [I] in the present invention.

The first method involves converting polysuccinimide to the following general formula (II
This is a method in which the derivative represented by [) is further bonded with a cytotoxic substance containing an amino group or an imino group in the molecule, and if necessary, reacted with a water-soluble aliphatic primary amine.

The second method is an effective method for binding cytotoxic substances having an amino group, and is a method that uses a functional group-donating compound (NH, -WS -Z
) and water-soluble aliphatic primary amines are both directly converted into succinimide group mido derivatives [I) using their amino groups.

As the first method, there are methods (1) to (3) below.

(11(a) Polysuccinimide [■] and (a) General formula □H2N W-8-Z (W tZ C same as above)
A compound represented by (b) is reacted with a water-soluble aliphatic primary amine if necessary, and then treated with an aqueous alkaline solution to obtain an intermediate of general formula (11).

(b) Then (c) a method of reacting a cytotoxic substance containing an amino group or an imino group with a water-soluble aliphatic primary amine if necessary (2) polysuccinimide and (a) ((J General Formula H2N-W-5-5- and [W is the same as above. Or an alkyl group (e.g. lower alkyl group having 1 to 5 carbon atoms), an aralkyl group (e.g. unsubstituted or substituted phenylalkyl group) or an aryl group (e.g. unsubstituted or substituted phenylalkyl group) Substituted or ゛represents a hydrocarbon group such as a substituted phenyl group)], (0) If necessary, after reacting with a water-soluble aliphatic amine, it is treated with an aqueous alkaline solution to form the general formula (m) (z = - 8-R) intermediate is obtained.

(b) Thereafter, (c) the cytotoxic substance containing an amino group or an imino group is reacted with a water-soluble aliphatic primary amine, if necessary.

After the reaction of raw (al or (b)), if necessary, the disulfide group of the acid is reacted with a thiol compound or a borohydride compound, etc., and reductively cleaved to form a polysuccinimide p in which 2=hydrogen atom. Derivative [I] is obtained.

(3) There is a method in which the thiol-type reactive polymer (Z-hydrogen atom polysuccinimide derivative [I) obtained by any of the above methods is reacted with an active disulfide compound. At this time, a polysuccinimide derivative [I] having z lle z/ is obtained.

Furthermore, as methods (1) and (2) above, (a)
el in (a), (b) and (b) in ")"
), to) are carried out sequentially in any order, a sequential reaction method,
There is a simultaneous reaction method in which two components are reacted simultaneously.

As a reaction solvent, DMF or DMSO, which is a good solvent for polysuccinimide, is used in the reactions (a) and (b).

Polysuccinimide (n) in DMF or DMSO at 1
~30(W/W)% how much it dissolves, (
(b) - to two components (0.001 to 10 mol per component per 1 mol of succinimide group, which is a monomer unit of polysuccinimide (IF)), and -40 to 8
It is appropriate to carry out the reaction under stirring at 0°C for 1 minute to 2 days. Unreacted components can be removed by dialysis or gel filtration. In order to obtain the intermediate represented by the general formula (III), it is desirable that the reaction intermediate in (11, +21) before the alkali aqueous solution treatment has an unreacted succinimide group. This is because it undergoes decomposition to form a -COOH group.

Carboxyl groups can be easily introduced into the thus obtained polysuccinimide derivative by subjecting it to alkaline conditions. Reaction with alkaline aqueous solution is DMF, DMS
This can be carried out by dropping an alkaline aqueous solution into the O solution or by adding a polysuccinimide derivative to the alkaline aqueous solution. The alkaline aqueous solution may have a P)I of 8 or more, but a 0° OIN to 2N NIOH aqueous solution is suitable.

The reaction of the thus obtained intermediate represented by general formula (II) with rXI in methods (1) and (2) can be carried out in a sequential reaction method in any order, or in a simultaneous reaction method in which it is carried out immediately. For the purpose of the present invention, it is preferable to introduce a large amount of (c), so it is preferable to react (c) after reacting (c).

The reaction is usually carried out in a homogeneous reaction system using water or an organic solvent such as DMF or DMSO as the reaction solution.

In the reaction, for example, a polysuccinimide derivative having a carboxyl group and a cytotoxic substance having an amino group or an imino group are reacted in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or dicyclohexylcarbodiimide. There are methods such as allowing them to coexist, and methods in which they are activated in the form of a polymer and reacted with a cytotoxic substance having an amino group or an imino group. The reaction temperature is 0℃~I
The appropriate reaction time for 00'C1 is 10 minutes to 10 days.

The ratio of the intermediate to be used is 10 to 200% of the intermediate.
'16 special D 100-2ooIs is desirable. If a carboxyl group remains unreacted in the reaction with pi, the carboxyl group is eliminated in the reaction with ni).

When using a cytotoxic substance containing an amino group, it can be produced by the second, simpler method.

A cytotoxic substance containing an amino group refers to a compound that is cytotoxic and has an amino group in the molecule containing a succinimide group of polysuccinimide.

In other words, it must have an amino group that reacts with a succinimide group to form an amide bond.

Examples of these include daunomycin, adriamycin, P-amino-N-[p-bis('2-10loethyl)aminocophenyl-3-hydroxyamide, 2-amino-N-[P-bis(2-chloroethyl) ) Aminocophenyl-3-hydroxy-2-hydroxymethylpropionamide, p-(bis(2-ri).

Examples include those having 7 aliphatic amine groups such as loethyl)amino]-L-phenylalanine.

As the second method, there are the following methods (41 to (6).

(41 polysuccinimide (ff) and (a) cytotoxic substance having an amino group (b) general formula H2N W 5-Z (
A method for obtaining a compound represented by (W, Z are the same as above) and e1 by reaction with a water-soluble aliphatic primary amine if necessary.

(5) Polysuccinimide (II) and (a) Cytotoxic substance having an amino group (b) General formula H2N-WS −
A method of obtaining a disulfide type polymer by reacting a compound represented by 5-R (W, R are the same as above) and e → with a water-soluble aliphatic primary amine if necessary, or a subsequent reaction if necessary. A method of obtaining a thiol-type polymer by reacting the disulfide group of the product with a thiol compound or a borohydride compound and reductively cleaving it.

(6) There is a method in which the thiol-type reactive polymer (polysuccinimide derivative [I] having z and L hydrogen atoms) obtained by any of the above methods is reacted with an active disulfide compound. As a result, z...
A polysuccinimide derivative [I] which is z' is obtained.

Furthermore, methods for (4) and (5) above include a sequential reaction method in which the reactions with each of the components (i), (b), and (e) described above are performed sequentially in any order, and reactions with multiple components are performed simultaneously. There is a simultaneous reaction method. Furthermore, when a reaction with component (c) is required, a two-step method is possible in which the reaction with one arbitrary component is carried out alone and the reactions with the remaining two components are carried out simultaneously.

As a reaction solvent, DMF or DMSO, which is a good solvent for polysuccinimide, is used in the first reaction and simultaneous reaction in the sequential reaction or two-step reaction. The second and subsequent reactions in a sequential reaction or two-step reaction can be carried out in an aqueous solvent when DMF or DMSO or the polysuccinimide derivative obtained in the first or second reaction becomes water-soluble. can.

In the reaction methods (4) and (5) above, the steps (a), (b), and reacting the Risaki components are carried out by the following operations.

Polysuccinimide (I[] or the unreacted succinimide group derivative after the first or second reaction, DMF, DMSO or water ~30 (W)
/W)% of the components to be dissolved and reacted ((l
, (0), one or more of No. e (0.001 to 10 moles per component per mole of succinimide group, which is a monomer unit of polysuccinimide (IF))
The mixture is reacted with stirring at -40 to 80°C for 10 minutes to 20 days. Unreacted components can be removed by dialysis or gel filtration.

The desired polysuccinimide derivative [I] can be obtained by distilling off the solvent under reduced pressure or by freeze-drying.

The molecular weight is controlled by selecting the condensation conditions to obtain polysuccinimide (I), and the separation of the polymer having the desired molecular weight is carried out by adjusting the D of polysuccinimide (II).
Molecular weight fractionation by gel filtration in an MF solution and molecular weight fractionation by gel filtration in an aqueous solvent after obtaining the polysuccinimide derivative [I].

In the method (4), 2 is preferably a hydrogen atom.

In order to improve the yield of the desired molecular weight fraction, the condensation conditions for obtaining polysuccinimide (n) are selected and the molecular weight fractionation is performed in advance by gel filtration with a DMF solution of polysuccinimide.

Thiol-type reactive polymer (polysuccinimide having 2-hydrogen atoms) according to methods (3) and (6) above
The active disulfide compound used in the method for obtaining a reactive polymer having an active disulfide group (z * z' polysuccinimide [■]) by reacting an active disulfide compound with an active disulfide compound is It is a disulfide of a group (2') covalent with the atom [which can form an active disulfide bond, and specific examples of such active disulfide compounds include disulfides of the aforementioned groups exemplified as 2', such as 2-pyridyldisthio 4-pyridyldisulfide, which is a disulfide of the group, 5,5'-dithiobis(2-nitro), which is a disulfide of a trophenylthio group.

The reaction between the thiol-type reactive polymer and the active disulfide compound is usually carried out in a homogeneous reaction system using water or an organic solvent such as DMF or DMSO as a reaction solvent. Or again,
The reaction can also be carried out using a reaction system in which an active disulfide compound or its acetone solution, dioxane solution, etc. are added and mixed to an aqueous solution of the polymer. Suitable reaction temperature is -5 to 70°C and reaction time is 1 minute to 24 hours.

In the complex of a cytotoxic substance and a bioactive protein that is the object of the present invention, the bioactive protein refers not only to a bioactive protein that has the ability to specifically bind to a tumor site, but also to a bioactive protein that has the ability to specifically bind to a tumor site. It also means a thing, a fragment.

Desirable physiologically active proteins in the present invention include, for example, fibrinogen and urokinase, which are known to specifically bind to thrombus sites, and immunoglobulin, which can specifically bind to specific antigens such as tumor cells, and specific Examples include lectins that specifically bind to the sugar structure of

Fibrinogen and urokinase are known to accumulate at tumor sites because there are many thrombotic sites around tumor cells. In addition, fibrinogen contains thrombin and C.
, 2+ to form a strong fibrin clot (cl
Fragment E, which is obtained by producing ot ) and further treating with plasmin, is also known to have the ability to specifically bind to thrombus sites.

Desirable bioactive proteins are immunoglobulins that can specifically bind to specific antigens such as tumor cells.

AFP and CEA are immunoglobulins.

HCG,) Immunoglobulin that can specifically bind to a specific protein, such as transferrin receptor, or target cells such as tumor cells or specific lymphocytes, or tissues containing them, obtained as an antigen. can be mentioned.

To obtain immunoglobulin, humans, monkeys, horses, cows, goats, sheep, rabbits, guinea pigs,
Using antiserum isolated from animals such as hamsters, rats, and mice, we use ethanol fractionation, ammonium sulfate fractionation, ion exchange, or molecular methods, or antibody-producing cells collected from animals immunized with antigens. Methods can be implemented in which hybridomas are obtained by making them cancerous with a substance or fused with myeloma cells, and monoclonal antibodies produced by these hybridomas are obtained.

Immunoglobulins have the ability to specifically bind to antigens even if their Fc portions are removed. In general, when immunoglobulin is digested with proteolytic enzymes such as papain, trypsin, chymotrypsin, and plasmin, so-called Fab fragments with one antigen-binding site are obtained, and if pepsin and trypsin digestions are performed under specific conditions, antigen-binding sites are obtained. A so-called F (a b')2 fragment having two is obtained.

When immunoglobulin is reduced with dithiothreitol, mercaptoethal, etc., a monomer having a mercapto group is obtained. When F (ab')2 is similarly reduced, Fab' having a mercapto group can be obtained.

The polysuccinimide derivative [I) and the physiologically active protein may be bound to each other by any of the known methods, such as those described below.

The first method is to use a mercapto group in a physiologically active protein having a mercapto group (for example, an immunoglobulin or its fragment F (ab')z which is reduced with dithiothreitol, mercaptoethanol, etc. or Fab'). or by introducing a mercapto group into the amino group of a physiologically active protein via a crosslinking group, and using the introduced mercapto group, the reactivity of z-2' in the polysuccinimide derivative [I) can be achieved. This method involves reacting with a polymer.

Many crosslinking agents are already known as crosslinking agents for introducing mercapto groups into amino groups of physiologically active proteins via crosslinking groups. The crosslinking agent used in the present invention may be any of such known crosslinking agents, but for example, the general formula [■1] H H5"z-CQl (I[a] (where L□
is a divalent organic group, and Q is the alcohol residue of imidoester.
For example, HCl salt, HBr salt, etc.) can be used.

In the above bifurcated formula [■3], the organic group represented by L is, for example, a linear or branched alkylene group having 2 to 6 carbon atoms such as ethylene or trimethylene; Examples of alcohol residues include alkoxy groups having 1 to 3 carbon atoms such as methoxy and ethoxy. More specifically, the above crosslinking agent [■1]
For example, methyl-3-mercaptopropyl N
HφHC1! I57'-) (H5(I(2-CH,C0CJ(3)
, methyl-4-mercaptobutyrimidate.

It can also be N-acetylhomocystine thiolactone.

Furthermore, general formula [IIb) Z' S-5-Lnee C-Q2[I[b]
and are as described above. Q2 represents the alcohol residue of the active ester. ] A compound represented by the general formula [1[[, ]% formula%] [wherein Z', L and Q are as described above.

] A compound or a salt thereof (e.g. H[I! salt, HBr salt, etc.), general formula (I[d)-←5-Ll-C
-Q□)2[:]na) [wherein L and Q□ are as described above. ] or a salt thereof (e.g. HCl salt,
HBr salt, etc.), general formula Cme)''-5-Ll-CQ
2), l (II[e) [wherein L and Q2 are as described above]. ] A compound or a salt thereof (for example, H[I! salt, HBr salt, etc.), S-acetylmercaptosuccinic anhydride, etc. can be used as a crosslinking agent. In this case, the amino groups of the physiologically active protein are reacted with a crosslinking agent, and then reduced using a reducing agent such as dithiothreitol or mercaptoethanol, or treated with an alkali to crosslink the physiologically active protein. A mercapto group can be introduced via the group. - Examples of the crosslinking agent represented by the above general formula (Ilrb) include N-succinimidyl-3-(2-pyridylthio)propionate; examples of the crosslinking agent represented by the general formula (Illc) include, for example, 3 Examples of the crosslinking agent represented by the general formula (md) of -(4'-dithiopyridyl)mercaptopropioimidate include dimethyl-3,3'-dithiobispropiodine, H[I! Inimidate [÷S CH2CH, C0CR3),
Examples of the crosslinking agent represented by the general formula [1[e] include dithiobis(succinimidylprodizuccinimidyl (N,N'-di/acetylpho), etc.).

The second method is to first introduce a 2' group into the mercapto group of a physiologically active protein having a mercapto group to form an sz' group, or to introduce -S to the amino group of the physiologically active protein via a crosslinking group.
This is a method of introducing a Z' group and then reacting this one SZ' group with polysuccinimide (1) in which 2 = hydrogen atoms.

A method for introducing a -sz' group into a physiologically active protein is to react a physiologically active protein having a mercapto group with an active disulfide compound, or to introduce a physiologically active protein into which a mercapto group is introduced into an amino group via a crosslinking group (the above-mentioned method). (see method 1) with an active disulfide compound;
There is a method of directly introducing a -8z' group via a crosslinking group by reacting with a crosslinking agent such as I[c).

The third method utilizes a mercapto group of a physiologically active protein having a mercapto group, or introduces a mercapto group into the amino group of a physiologically active protein via a crosslinking group, and utilizes this mercapto group to Active proteins or bioactive proteins with mercapto groups introduced and polysuccinimide derivatives which are 2-hydrogen atoms [! ] and are bonded together using a crosslinking agent. As the crosslinking agent, a bifunctional crosslinking agent capable of reacting with a mercapto group is used.

Examples of suitable bifunctional crosslinking agents capable of reacting with mercapto groups for this purpose include simaleimide compounds represented by general formula (IV), benzoquinone, and the like.

[L represents a divalent organic group. ] Examples of the divalent organic group represented by LIA include a phenylene group, an azobenzenediyl group, and a group having the general formula -((I(,), -0(CH2)a-λ
2 [In the formula, tameyo and 12 are integers from 1 to 3.

] Examples include an alkylene group via an oxygen atom, which is formed by the following.

More specific examples of simalimide compounds include N,
Examples include N'-(1,2-phenylene) dimalephenylene) simaleimidoylamino) azobenzene bis(N-maleimidomethyl) ether.

In order to bond a physiologically active protein and a succinimide derivative [I] by the method of Suihiro 3, ■ A biologically active protein having a mercapto group or a biologically active protein into which a mercapto group has been introduced ■ Polysuccinimide which is a Z-hydrogen atom Derivative (I
') with an excess of crosslinking agent relative to the mercapto groups it has, and then react with the other.

The fourth method utilizes the amino group of a physiologically active protein to introduce a functional group capable of reacting with a mercapto group into the physiologically active protein via a crosslinking group, and then introduces a polysuccinimide derivative, which is a Z-hydrogen atom. This is a method of reacting with [I].

Examples of the functional group capable of reacting with a mercapto group include a maleimide group represented by the formula, and the general formula CHL [wherein L is a halogen atom]. ] Examples include a halogenated methyl group represented by the following. Examples of the halogen atoms include chlorine, bromine,
Examples include an iodine atom, but an iodine atom is preferred.

Crosslinking agents that introduce functional groups through the amino group ξζ of physiologically active proteins and crosslinking groups are already known, but among them, preferred crosslinking agents include those of the general formula (VJ [wherein L3 is 2 and Q2 is as described above.]

In this ξ, the divalent organic group is, for example, a phenylene group,
General Formula % Formula % [In the formula, A is a phenylene group or a cycloalkanediyl having 5 to 7 carbon atoms, and b is an integer of 1 to 3. ] Examples include a group represented by the following formula, an alkylene group having 2 to 6 carbon atoms, and the like.

As a specific example of the crosslinking agent represented by the above general formula (V), for example, m-maleimidobenzoic acid N
- N-hydroxysuccinimide ester of hydroxysuccinimide ester (boxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester of carboxyphenylmethyl)maleimide maleimide cabroxy)succinimide ester imide ester above in Section 1.2.3 In method 4 and 4, the reaction between the amino group of the physiologically active protein and the crosslinking agent can be carried out as follows.

P prepared by dissolving physiologically active proteins to a concentration of 0.5 to 100 η/rnl (preferably 1 to 20 η/rnl)
1 to 100 mol of a crosslinking agent per 1 mol of physiologically active protein is added to a buffer solution of H5 to 9, and the mixture is reacted at 0 to 50°C with stirring.

The reaction time depends on the reaction scale and reaction conditions, but is usually within 2 days. The crosslinking agent is used as an aqueous solution or, if the crosslinking agent is not soluble in water, a small amount of an organic solvent (eg DMF, DMSo).

It is used as a solution dissolved in 1,2-dimethoxyethane, 1,4-dioxane, methanol, ethanol, acetone, etc.).

After completion of the reaction, low molecular weight substances can be removed and purified by conventional means such as dialysis or gel filtration.

The reaction in which the physiologically active protein having a mercapto group or introduced therein and the thiol-type vorizaxinimide derivative in the third method are bonded using a bifunctional crosslinking agent can also be carried out in the same manner as above. can.

In methods 1 to 4 above, the final reaction to obtain a complex of a cytotoxic substance and a physiologically active protein is performed using a physiologically active protein, a physiologically active protein into which a mercapto group has been introduced via a cross-linking group, or a modified product thereof (hereinafter referred to as , these are collectively referred to as reactive physiologically active proteins) and the polysuccinimide derivative [I) in an appropriate combination as explained in Methods 1 to 4 above, and are reacted in the following manner. urge

The reactive physiologically active protein is added to a buffer solution with a pH of 5 to 9 at a concentration of 0.5 to 100' f/m/ (preferably 1 to 20
~/-) and an aqueous solution of the polysuccinimide derivative [I) to be bound to the reactive physiologically active protein or the same buffer solution as above, and mix the solution to give a concentration of 0 to 50 Allow to react by standing at ℃ or stirring gently. The polysuccinimide derivative (1) is 0.1 to 70% per mole of physiologically active protein.
Use moles. The reaction time is usually within 3 days.

Separation and purification of the target complex from the reaction mixture can be carried out by gel filtration, affinity chromatography, dialysis, etc.
L-aspartic acid 66 using common techniques such as precipitation
．． 85% phosphoric acid 20.6- was added to 5F and heated to 190℃ using a rotary evaporator while heating at 10m for 5.5 hours.
Polymerization was carried out under reduced pressure of Htt.
After polymerization, DMFI was dissolved and reprecipitated with water. After reprecipitation, it was filtered through a glass filter and vacuum-dried at 80° C. for 3 days to obtain polysuccinimide.

The obtained polymer was measured for infrared absorption using the KBr tablet method, and the results were 1800 and 1720.

At 1660 as, absorption peculiar to the succinimide group was observed.

Molecular weight measurement was performed by GPC measurement.

GPC collar A for DMF (AD-80M/S-Showa Denko)
The obtained polysuccinimide was diluted with DMF +0.
It was dissolved in a Q1MI4Br solvent to a concentration of 0.2H, 250 μl of the solution was injected, and the same solvent was used as an elution solvent for measurement with a differential refraction needle. A calibration curve was created using polyethylene glycol with a different molecular weight. The obtained polysuccinimide had a weight average molecular weight of 8°9X10' and a number average molecular weight of 1°5X10 in terms of polyethylene glycol.
'Met.

Reference Example 2 Polysuccinimide 1.5611 (obtained in Reference Example 1)
(corresponding to 15 mmol of succinimide group) to 90-
Dissolved in MF.

This solution contains 34.7 μ (0.45 mmol) of cysteamine.
), ethanolamine 6531”P (10,7mm
Add a solution of ol) mixed with DMF IQ-,
The reaction was carried out at 4°C for 24 hours. After the reaction, add 1N Na to the reaction solution.
Approximately 50 ml of OH solution was added dropwise over 1 hour with stirring.

After that, dialysis was performed with an aqueous solution having a pH of 4. X4 with Diaflow membrane ■ (manufactured by Amicon, molecular weight 10,000 cut)
Gel filtration was performed using a 0aa column to remove low molecular components. Freeze-drying was performed to obtain an intermediate having the general formula (II[) and having a 2-hydrogen atom.

When the thiol group in the intermediate was determined by the method shown in Reference Example 4 described later, it was found that 1°5×10
'moles of thiol groups were present. The amount of carboxyl group was determined by acid-base titration according to a conventional method. As a result, 2°8×10 −8 moles of carboxyl groups were present in intermediate lf. No unreacted succinimide group was observed from IR and 13 C-NMR measurements.

From the above measurement results, the amount of cysteamine introduced per mole of succinimide group (X mole) and power.

The amount of introduced carboxyl group (7 moles) was determined from the following simultaneous equations.

Here, a, b, c, t, and s are as follows.

Molecule of the monomer unit in which the succinimide group reacted with ethanolamine 1ta-158 Molecule of the monomer unit in which the succinimide group reacted with cysteamine it
b = 174 Molecular weight of monomer unit with hydrolyzed succinimide group C = 115 11t1 body (III31 The amount of cysteamine introduced in f is 1.42 x 10 molar intermediate (nI) 1 The amount of carboxyl group introduced in f/ The result of the molar calculation of S depth 2°8X10 was X -0.02 mole, yP=o, a mole.

The molecular weight was determined by measuring the terminal amine. 5 wv of the above intermediate was dissolved in 0° IM sodium borate solution 1 and then 2.4.6-)linitrobenzenesulfonic acid (P
Add 0.1 mZ of a solution of TNBS (abbreviated as TNBS) dissolved in 10-0° IM sodium borate solution,
After the reaction was carried out at 25°C for 45 minutes, 0°C of 1M sodium borate solution was further added and the absorption at 4200 m was measured. A molecular extinction coefficient of 11.100 was obtained from a calibration curve using a known amount of aspartic acid, and the value of the terminal amine was calculated using this molecular extinction coefficient. As a result, 5×10′ moles of amino groups were present in intermediate lf. From this result, the average molecular weight is approximately 20.000.

``From the value of t7, the molecular weight of the average monomer unit is 145.
．． 4, and the degree of polymerization is n'0m+/'+r' from the molecular weight.
+P'+(1'(7) Average value c138゜"eV n
The average value of '-1-m' is 41. The average value of r'+r' is 94. The average value of P+9' is 3.

Reference Example 3 20 μ of the intermediate obtained in Reference Example 2 was dissolved in 5-water,
To this was added 6811v of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and I N HC/
The pH was adjusted to 5.0. A solution of 20 η of daunomycin hydrochloride dissolved in 31 nl of water is added dropwise to this solution.

After reacting for 1 hour at room temperature, the pH was adjusted to 9.5 with IN NaOH. Then 0°15NNacI! 5ephadex G-25 Superfine (manufactured by Pharmacia) equilibrated with aqueous solution 3.9 aag X7°0a1
After gel filtration was performed using column 1 to remove low molecular components, 8% NaCl! After dialysis with an aqueous solution, ethanolamine hydrochloride 100F was obtained.

Add 200 μl of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and add 1NHCl! pH at
5.0 and the reaction was carried out at room temperature for 1 hour. Then 0. β
%NaCl! After dialysis with an aqueous solution, freeze-drying was performed.

Yield: 18η The amount of daunomycin introduced into the obtained polysuccinimide derivative was 7〒忰Q, IM sodium phosphate-〇. 1
485 n in a solution of 5M saline buffer CP117.4)
It was determined from the absorption of m. 49×10′ moles of daunomycin were incorporated into 1 g of polysuccinimide derivative.

The thiol group was quantified by the method of Reference Example 4 described below. 9.8X10~5 in polysuccinimide derivative lf
There were moles of thiol groups.

No unreacted carboxyl group was detected by iR and C-NMR.

As a result of the above, 0,002 moles of thiol group and 0.1 mole of daunomycin are bound per mole of succinimide group.

Quantification of terminal amino groups was carried out in the same manner as in Reference Example 2. 3゜53X10' in polysuccinimide derivative lf
moles of amino groups are present and the average molecular weight is 28,00
It becomes 0.

From these values, the average value of the degree of polymerization n+m+/+r+p+q is 13B, the average value of n0mO) is 14°/the average value of +r is 12
1. X-daunomycin, 2 with an average value of p1q of 3
- It was found that a polysuccinimide derivative [I] containing hydrogen atoms was obtained.

Reference Example 4 This example uses thiol type intermediate (1) (Z=hydrogen atom)
The present invention shows a method for converting 1 to an intermediate [I] between z and z', and also shows a method for quantifying the thiol group in the 1,000-ol type intermediate [I].

Intermediate (III) 2 Q 119 obtained in Reference Example 2
3-0.1M sodium phosphate-1mM EDTA
(PH7.0), dithiothreitol 10w9 was added, and a reduction reaction was carried out at 30°C for 1 hour. 5eph*dex■G equilibrated with the above buffer solution after reaction
-2'57. - Par 7, I, N (manufactured by Ffurumasha)
2.0anXX 15cm Tegel filtration was performed to remove low molecular weight components, and then diluted 20 times with buffer. To 500 ul of this reaction solution was added 20 ttl of a methanol solution of 5 mM 4-pyridyl disulfide and allowed to react for 15 minutes at 30°C. Huain (manufactured by Fufurumasha) 2.0 alOXl, 5
[In II, the gel was treated and separated into a high molecular component and a low molecular component.

4-thiopyridone compounded in the low molecular component is 324 n
Measured in m. Molecular extinction coefficient of 4-thiopyridone: 1.9
When the amount of thiol groups in intermediate [I] (Z-hydrogen atom) was calculated using 8 x 10', it was found that 1°4 x 10 mol of thiol groups were present in intermediate lf.

Reference Example 5 86 mm of polysuccinimide obtained in Reference Example 1 (0.89 mmot of succinimide groups) was dissolved in 2 mm of DMF.

Jin's liquid number zeta daunomycin hydrochloride 20v9 (35μmo
l ) and then 12μI of triethylamine! (86
μmol) was added and allowed to react at room temperature for 10 days. Then cysteamine 6°7 cape, ethanolamine 0.13v
A mixed solution of DMF 1- was added and reacted at 4°C for 4 hours.

After dialysis of this reaction solution for 2 days against a water bottle, the pore size was 0.45.
Remove the insolubilized portion with a μm membrane (Millipore filter) and remove the R solution with 5ephadex G-25 Super Fine (
)1 Lumasha Co., Ltd. 1l) 2.6 [Low molecular components were removed by gel filtration using a column of IIJ2'X403 using distilled water as an eluent.

Thereafter, freeze-drying was performed to obtain a polysuccinimide derivative. Yield: 401IP0 IR, 13cm NMR measurements showed that no unreacted succinimide group was detected.

The amount of daunomycin introduced per mol of succinimide group in the obtained polysuccinimide derivative is as shown in Reference Example 3.
was calculated using the same method.

1.2×・10− in polysuccinimide derivative lf
4 moles of daunomycin were bound.

The amount of bound cysteamine was determined by the method of Reference Example 4. There were 2.5×10′ moles of cysteamine in the polysuccinimide derivative lf.

No unreacted succinimide group was detected from IR and CNMR measurements.

From the above values, 1 mole of succinimide group is combined with 0.02 mole of daunomycin and 0.005 mole of cysteamine.

When the terminal amino group was quantified in the same manner as in Reference Example 2, 1°25×1
0'' moles of amino groups were present.The average molecular weight was s
o, ooo.

From the above results, the average value of the degree of polymerization n0m+/+r0p+9 is 478, the average value of n0m is 10, the average value of /+r is 466, and the average value of p1q is 2. It was found that a polysuccinimide derivative [I] in which daunomycin, ZI = hydrogen atom, was obtained.

Reference Example 6 Production method of immunoglobulin The production method of monoclonal antibody against human transferrin/core setter is as follows.
e 2864j-Ml 1980°.

Lung lymphocytes and non-immunoglobulin-producing myeloma cells of BALB/C mice immunized with strain 562 obtained from a human chronic myeloid leukemia patient; 4519415XXO,
BU, 1 strain was fused according to a conventional method, and fused cells producing antibodies against transferrin receptor were selected.

The above-mentioned fused cells were injected into BALB/C mice pre-administered with Blistane, and ascites fluid was collected about 2 weeks later.

Immunoglobulin was purified from ascites. Ascitic fluid was precipitated using a 50% saturated ammonium sulfate aqueous solution, dissolved in sodium phosphate buffer, and dialyzed.
After obtaining the fraction, further Amicon membrane (Amicon a[-PM-
It was concentrated in step 3) to obtain a solution with a protein concentration of 5η/-.

Example 1 Fibrinogen-Midori (purchased from Midori Juji) 10
55”P (fibrinogen amount 330 wf) in distilled water 1
0 mHm was added, and the mixture was stirred at room temperature for 30 minutes to dissolve. In this solution, dimethyl-3-3'-dithiobispropionimidate (purchased from Pierce) 121119 and dithiothreitol 60119 were dissolved in six bottles of distilled water and reacted at 23°C for 40 minutes. 0°OIM phosphate buffered saline (PB
S) 5ephadex G-2'5 super 7) p-in, (
Gel filtration was performed using a 2°6alOX 40cm column (manufactured by Lumasha, Inc.) to remove low molecular components. To this aliquot, 32cm of a reactive polymer having active disulfide groups obtained by subjecting the thiol-type reactive polymer obtained in Reference Example 3 to the same method as in Reference Example 4 was added and reacted at 4°C for 18 hours. I did it. Post-reaction stage - 5eph equilibrated with buffer
Gel filtration was performed using a 2.6 csX X 1 (jOam) column of arose CL-6-3- (manufactured by Pharmacia) to separate the fibrinogen fraction.

Protein recovery rate is 20%.

In order to confirm that the coagulation ability of fibrinogen had not changed in the obtained complex, the coagulation ability was measured by the method described in A. did. Fibrinogen in the complex 0.56 cape/-
Adjust the aqueous solution so that
0.54 of the same buffer solution was added, and after leaving at room temperature for 1 hour, the resulting clot (CLet) was removed, and the fibrinogen concentration of the remaining solution was measured by measuring the absorbance at 280 am, and the coagulation rate of fibrinogen was calculated. The coagulation rate was 85%.

The coagulation rate of the fibrinogen used was 86'%, and it was confirmed that the coagulation ability of fibrinogen did not decrease even when it was formed into a composite. Absorption at 280 nm and 4
The number of daunomycins bound per fibrinogen molecule was calculated from the absorption at 85 nm, and 40 daunomycins were bound to one fibrinogen molecule.

The buffer solution was exchanged on a 5ephadex G-25 (7, manufactured by Lumasha) column obtained in Example 6 and equilibrated with cloflin 31%, and the buffer was exchanged at 5vV/ -To the solution 6- condensed by 1n to 6-, add 30- of N-hydrochelesuccinimide ester me4Q mM dioxane solution of N-(4-carboxycyclohexylmethyl)maleimide to 30°C.
A time reaction was performed. After reaction - use buffer solution 8ep
hadex■c-252-perfine (2) (manufactured by Lumasha) 2. The gel was filtered with an Ocrnlf X20α column to remove low molecular weight components.

30cm of the reactive polymer obtained in Reference Example 3 was added to buffer solution A3-
13 kg of dithiothreitol was added to this solution and reacted for 2 hours at 30°C, followed by gel filtration in the same manner as above to remove low molecular components.

Both fractions after gel filtration were mixed and allowed to stand at 4°C for 18 hours to react. In order to remove unreacted reactive polymers, the reaction solution was treated with Protein 80 (manufactured by Farhesha) 30.
- applied to a column equilibrated with 0.1M phosphate buffer (pH 8,20), and after thoroughly removing non-adsorbed components with equilibration buffer, protein was added to 0.1M phosphate buffered saline (pH 5,8). The components adsorbed on the A[F] column were eluted.

The eluate was dialyzed against 0.1M phosphate buffer (PH8, 0>II!). After dialysis, the dialysis solution was concentrated with an Amicon membrane (PM-10, manufactured by Amicon) to a protein concentration of 1. A solution 6- of .13"F/- was obtained. The number of daunomycin bound per immunoglobulin molecule was calculated from the absorption of the obtained complex at 280 nm and 485 nm. 40 daunomycins were bound to it.

To confirm that the immunoglobulin complex retains the antigen-binding ability as an antibody, the immunoglobulin was iodine-labeled using chloramine.

In a plastic tube, add 5 μl of 0°5M sodium phosphate buffer pH 7.5 (hereinafter referred to as buffer B), Na
Add 126I to Q and 3mC1, stir, and then add 200μ
After adding 20 .mu.l of a buffer B solution of chloramine-T of f/ml and stirring for 20 seconds, 20 .mu.l of a 0.5 k/ml immunoglobulin buffer B solution was added and a reaction was carried out with stirring for 15 seconds.

After the reaction, 14 pfl/ml of Na2S20. (F) Buffer B solution 400μl, Kl of 20η/- Buffer B
25 μl of the solution and 25 μl of a 5% BSA buffer B solution were added. Thereafter, gel filtration was performed using a 5 ephadex G-25 (7) column using buffer B as an eluent, and the protein eluate was fractionated to remove low molecules.

In order to determine the specific radioactivity of the 1261-labeled immunoglobulin thus obtained, the protein concentration was determined from the absorption at 280 nm, and the total radioactivity was measured, and the result was 4.3 mC1/■.
The specific radioactivity was . Add an amount of radioactivity of the above 126A-labeled immunoglobulin (2 x 105 cpm) to 1'0% FC8-RPM ÷ -1640 medium (purchased from Hani Chemical), and add 1 volume of the above complex to 0.1 volume of this solution. Add 0.4 - of lysed Ram's solution to give a concentration of .0 to 0.1'f/-, and add CCRF-C1M cells (which are known to have human transferrin receptors) to this solution.
Di X 10' cells/- 10chi FC5-R
Add PM+-154Q medium solution 0.5- and heat at 4°C.
Incubation was carried out for hours. After this 2.00O
Centrifuge at rpm for 5 minutes, discard the supernatant, and collect the precipitate.
After adding 5% BSA-Hank's buffer, centrifugation was performed at 2.000 rPm for 5 minutes, the upper groove was discarded, and the radioactivity of the precipitate was measured.

For comparison, an experiment was conducted in which immunoglobulin was added at the same concentration instead of the complex, and it was found that the complex and immunoglobulin were 126% per CCRM-CEM cell.
■ The effect of inhibiting the binding of the labeled immunoglobulin was the same, indicating that the immunoglobulin complex had the same binding ability as the original immunoglobulin.

Example 3 2゛The immunoglobulin obtained in Reference Example 6 was prepared in the same manner as in Example 2.
0°IM phosphoric acid MN solution containing mMEDTA (M street solution A)
2Q m to 6rnl of the solution made 551ny/'-
Add 30 μl of ethanol solution of MN-succinimidyl-3-(2-pyridylthio)propionate and incubate at 23°C.
Incubated for 30 minutes. After the reaction, 5 kg of glycine as a reaction terminator and 5 kg of dithiothreitol as a reducing agent were added and incubated at 23°C for 30 minutes. 5ephadex@c, -25(7
Gel filtration was performed using a 2°0 [IOX 20-port column (manufactured by Sha Co., Ltd.) to remove low molecular weight components, and protein fraction 7- was fractionated. Reactive polymer 1 having active disulfide bonds obtained by applying the same method as in Reference example 4 to the thiol-type reactive polymer obtained in Reference example 3 to this fractionated liquid
0~ was added and incubated at 4°C for 18 hours. Thereafter, in order to remove unreacted reactive polymers, the mixture was separated and concentrated using a Protein A column in the same manner as in Example 2, and a protein concentration of 1.12''/- was obtained.

The number of daunomycins bound per immunoglobulin molecule was calculated from the absorption at 2800 m and 485 nm of the obtained complex, and it was found that 40 daunomycins were bound to one molecule of immunoglobulin.

Confirmation of whether the immunoglobulin in the complex retains the ability to bind to the antigen as an antibody is carried out in the same manner as in Example 212
When the ability of the 61-labeled immunoglobulin to inhibit binding with the antigen was compared with that of the original immunoglobulin, it was confirmed that the binding ability was not decreased.

Example 4 Immunoglobulin 1.2f was dissolved in 0°1M acetate buffer (PH4
, 5) Dissolve in 40- and add 24 μ of pepsin to 3
After decomposition at 7°C for about 18 hours, the decomposition products were applied to a 3°53y x 140 column of 5ephadex c-200 (manufactured by Fufurumasha) in physiological saline to remove proteins that had a molecular weight of about 100,000. Take out, F (a
b') a was obtained.

1.141 nl of F (ab') 210 myQ mM phosphate M solution pH 6.5) with 0.4 N-hydroxysuccinimidyl-m-maleimidobenzoic acid
0.05- of N,N-dimethylformamide containing 2 capes was added and reacted at room temperature for 40 minutes. 0.14 after reaction
5 m containing M sodium chloride, l m M-EDTA
Dialyze against M acetic acid R buffer pH 5.5 to remove excess reagent.
-maleimidobenzoyl F (ab')2 was obtained.

The reactive polymer 41 obtained in Reference Example 3 was dissolved in 0° 1M sodium phosphate-1mM EDTA buffer (PH 7,0) 3
-, add 13 kg of dithiothreitol to this solution, react for 2 hours at 30°C, and then add 5 mM acetate buffer pI (5,
2.0 cm 0 x 13 of 5ephadex G25 Suha Fine (manufactured by Pharmasha) equilibrated with 5.
Gel filtration was performed using a 5 cm column to remove low molecular components.

M-maleimidobenzoyl F (ab') 230μ equivalent M loading solution and reactive polymer equivalent 15η buffer solution were mixed, and then 0.5M phosphate buffer (pH 6,5) 0.3 r
nl was added and reacted at 4°C for 16 hours.

5ephadex@G-20 with physiological saline after reaction
0()1 manufactured by Lumasha) 3.5 cm g X 14
A fraction with a molecular weight of 100,000 to 150,000 was collected using a 0 cm column.

The number of daunomycin bound per molecule of F (a b')2 was calculated from the absorption at 280 nm and 485 nm of the obtained complex.
Forty daunomycins were bound in one molecule. To confirm whether F (a b')2 in the complex retains the ability to bind to the antigen, use F (a b')2 as a comparison.
As in Example 2, the ability to inhibit the binding of 125 l-labeled immunoglobulin to the antigen was determined using the same method as in Example 2.
b') When compared with 2, (Fab'
) It was confirmed that the binding ability of 2 did not decrease even when it was formed into a complex.

Example 5 Immunoglobulin was digested with Pefcinte in the same manner as in Example 4. L/1'' (F ab')2 was obtained.F(ab')
0.1M Sodium Phosphate with 240 Capes - 1mMED
TA (pH 6,0) buffer (hereinafter referred to as buffer C) 8
．． 488! , 1 g of 0.1 M 2-1 captoethylamine in buffer C was added and the mixture was reduced at 87° C. for 90 minutes. After the reaction, the solution was equilibrated with buffer C for 8ep.
Low molecular weight components were removed by gel filtration using Hadex G-25 (manufactured by 7 Almasya). After separating the protein fraction, N, N
'-Q-phenylene dimaleimide 0.1M6M-1m
25.7 g of a saturated solution of MEDTM buffer pH 5.0 was added and reacted for 80 minutes with stirring. After the reaction, low molecular components were removed with 8sphadex G-25 equilibrated with 0.02M acetate buffer pH 5.0,
After that, the Fab into which the maleimide group was introduced was concentrated.
' To obtain a solution with a concentration of 2.51 F/sl, the reactive polymer 41η obtained in Reference Example 5 was mixed with 0.1 M sodium phosphate-1 m MED buffer (puy, o) 8
ml and add dithiothreitol 18p to this solution.
Add I and react at 80°C for 2 hours, then add 5mM acetate buffer containing 0.14M sodium chloride-1mM-EDTA pH
Gel filtration was performed using 8ephadex G-25 superfine equilibrated for 5.5 times to remove low molecular components.

'Mix a buffer solution equivalent to Fab'15q into which a maleimide group has been introduced and a buffer solution equivalent to 15 da reactive polymer, and then
．． Add 5M phosphate buffer (PH6,5) 068 resistant at 4°C.
The reaction was carried out for 16 hours. After reaction, saline solution 8ephad
ex G-200 () 1 Lumasha GS) 8.5 a
Both fractions, equivalent to more than 200,000 globular proteins, were fractionated using an nφX140an column.

The amount of bound daunomycin per Fab' molecule was calculated from the absorption at 485 nm and 280 nm of the resulting complex, and it was found that 10 daunomycins were bound per Fab' molecule.

In order to confirm the binding ability of Fab' to the antigen, the binding inhibition ability of the knee-labeled immunoglobulin to the antigen was measured in the same manner as in Example 2, and the binding inhibition ability was clearly observed.

Claims (1)

[Claims] (1) The following general formulas [1] to [6] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [1] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [2] ▲ Mathematical formulas, chemical formulas , tables, etc. ▼ [3] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [4] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [5] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [6] [Above In formulas [1] to [6], X, Y, W and Z
have the following meanings. X: Reactive residue of a cytotoxic substance having an amino group or imino group in the molecule Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group Z: Hydrogen atom or a group represented by the general formula Z' (however, ,Z
When is a hydrogen atom, the -SH group represented by -SZ can form an -S-S- bond in equilibrium with other -SH groups within or between molecules; shall be expressed. ) Z′: a group capable of forming an active disulfide bond with an adjacent sulfur atom] Consisting of the structural units represented by [1], [2], [3], [4], and each structural unit in one molecule. The numbers [5] and [6] are n, m, l, r, p and q, respectively, and these numbers are 0 or natural numbers, and the relationship between them is n+m≧2 0.001≦(p+q)/ (n+m+l+r+p+q)
≦0.50, and the average molecular weight is 2,000 to 1,00
A protein-polysuccinimide complex comprising a polysuccinimide derivative [I] having a molecular weight of 0,000 and a physiologically active protein. (2) The binding between the polysuccinimide derivative [I] and the physiologically active protein is
The protein-polysuccinimide complex according to claim 1, which is a disulfide bond formed by using an SZ group and a mercapto group of a physiologically active protein having a mercapto group. (3) The binding between the polysuccinimide derivative [I] and the physiologically active protein is
2. The protein-polysuccinimide complex according to claim 1, which utilizes an SZ group and an amino group of a physiologically active protein, and is bonded through a crosslinking group between them. (4) The binding between the polysuccinimide derivative [I] and the physiologically active protein is
2. The protein-polysuccinimide complex according to claim 1, wherein the SZ group and the mercapto group of a physiologically active protein having a mercapto group are bonded via a crosslinking group therebetween. (5) The protein-polysuccinimide complex according to claim 1, wherein the physiologically active protein is fibrinogen, urokinase, or immunoglobulin. (6) The following general formulas [1] to [6] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [1] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [2] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [3] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [4] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [5] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [6] [Above formulas [1] ~ [ 6], X, Y, W and Z
have the following meanings. X: Reactive residue of a cytotoxic substance having an amino group or imino group in the molecule Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group Z: Hydrogen atom or a group represented by the general formula Z' (however, ,Z
When is a hydrogen atom, the -SH group represented by -SZ can form an -S-S- bond in equilibrium with other -SH groups within or intermolecularly; shall be expressed. ) Z′: a group capable of forming an active disulfide bond with an adjacent sulfur atom] Consisting of the structural units represented by [1], [2], [3], [4], and each structural unit in one molecule. The numbers [5] and [6] are n, m, l, r, p and q, respectively, and these numbers are 0 or natural numbers, and the relationship between them is n+m≧2 0.001≦(p+q)/ (n+m+l+r+p+q)
≦0.50, and the average molecular weight is 2,000 to 1,00
In producing a protein-polysuccinimide complex in which a polysuccinimide derivative [I] having a molecular weight of 0,000 and a physiologically active protein is combined, a mercapto group of the physiologically active protein or a mercapto group newly introduced into the physiologically active protein is used. and a method for producing the protein-polysuccinimide complex, characterized in that the polysuccinimide derivative [I] is bound to a physiologically active protein using the mercapto group of the polysuccinimide derivative [I]. (7) Physiologically active proteins and polysuccinimide derivatives [
The method of bonding using the mercapto group of [I] is used to connect bioactive proteins with mercapto groups, or bioactive proteins in which a mercapto group is introduced into the amino group via a crosslinking group, and Z=
The protein according to claim 6, which is a method of reacting the polysuccinimide derivative [I] which is Z'.
Method for producing polysuccinimide complexes. (8) Physiologically active proteins and polysuccinimide derivatives [
The method of bonding using the mercapto group of [I] involves introducing a Z' group into the mercapto group of a physiologically active protein having a mercapto group to form a -SZ' group, or introducing a crosslinking group into the amino group of the physiologically active protein. -SZ' group is introduced through Z
7. The method for producing a protein-polysuccinimide complex according to claim 6, which is a method of reacting with a polysuccinimide derivative [I] in which = hydrogen atom. (9) Physiologically active proteins and polysuccinimide derivatives [
I] is a method of bonding using a mercapto group between a biologically active protein having a mercapto group or a biologically active protein in which a mercapto group is introduced into an amino group via a crosslinking group, and a biologically active protein having a mercapto group (Z = hydrogen atom). The protein-polysuccinimide derivative according to claim 6, which is a method of reacting one of the polysuccinimide derivatives with an excess of a crosslinking agent with respect to the mercapto group thereof, and then reacting with the other polysuccinimide derivative. Method for producing succinimide complex. (10) A method of bonding using the mercapto group of a physiologically active protein and a polysuccinimide derivative [I] produces a biologically active protein in which a functional group capable of reacting with the mercapto group is introduced into the amino group via a crosslinking group. 7. The method for producing a protein-polysuccinimide complex according to claim 6, which is a method of reacting polysuccinimide derivatives in which Z=hydrogen atom.