JPH11152234A - Bivalent reactive water soluble polymer derivative and complex containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject derivative having a reactive site for amino group, thiol group part and a potential thiol group part, and capable of preventing a reduction of the activity of a ligand and an effect to an acting material. SOLUTION: This bivalent reactive water soluble polymer derivative has a reactive site with amino group, a thiol group part, a potential thiol group part, and is preferably expressed by the formula (S is a thiol group part or a potential thiol group part; P is a water soluble polymer; N is a reactive site with amino group; R<1> , R<2> are each a linking group). Preferably, the potential thiol group is S-acetylthio group. The water soluble polymer is a biodegradable polymer such as a polyamino acid and a polyoxy acid, or a synthetic polymer such as a polyethylene glycol and a polyacrylamide, and preferably has 2,000-6,000 molecular weight. The reactive site with the amino group is preferably succinimide group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a heterogeneous compound in which a ligand (a protein such as an antigen or an antibody, a hormone, etc.) and an active substance such as a small molecule drug, a marker molecule, a protein, a micelle, or a liposome are bound as a spacer. The present invention relates to a bivalent reactive water-soluble polymer derivative, a complex obtained therefrom, a pharmaceutical / diagnostic composition containing the complex, and a method for producing the complex. The complex obtained in the present invention can be used for a pharmaceutical carrier, diagnosis, inspection, and the like.

[0002]

2. Description of the Related Art In recent years,
For drugs, isotopes, marker substances, etc., antigens, antibodies,
Research and development for imparting specificity by binding proteins such as transferrin and ligands such as hormones have been performed in various fields. In particular, it is expected that proteins such as antibodies are bound to drugs such as anticancer drugs and proteins such as radioisotopes and toxins and used as site-specific drugs or highly specific diagnostic agents. Further, a complex in which an antibody and an enzyme such as alkaline phosphatase are bound has also been put to practical use as an assay reagent or the like.

These complexes are formed by direct binding or via a carrier such as a liposome. When an antibody is used as an example, an active substance (enzyme, liposome, etc.) having a hydrazide group added by oxidizing the antibody sugar chain to an aldehyde. ), A method in which a maleimide group is introduced into the amino group of an antibody and a thiol group is introduced into an active substance to bind the active substance, a method in which an endogenous disulfide group is reduced with a reducing agent to generate a thiol, and a reaction with avidin / There is a method of binding via biotin (review, enzyme immunoassay, 3rd edition, Ishikawa et al., Medical School, 1987).

On the other hand, polyethylene glycol (hereinafter, referred to as polyethylene glycol)
It may be referred to as “PEG”. ) Is a flexible water-soluble polymer, and many examples have been studied as protein modifiers. PEG has low toxicity and low immunogenicity, and it has been known that the effect of PEG modification to significantly improve the retention of proteins in blood is known (see "DDS Advances 199").
5-1996 "Nakayama Shoten p82-94 (199
5)).

[0005] Recently, studies have been made on the use of this PEG as a spacer as described above. Particularly in the liposome field, research on antibody-bound liposomes using a PEG moiety as a spacer has been actively conducted. Also,
It is known that lipids having polar groups, amphiphilic polymers, and the like form fine particles such as lipid micelles, polymer micelles, liposomes, and lipid micelle closed double layers.

In recent years, many attempts have been made to use these fine particles as carriers for drugs and nucleic acids. Liposomes can enclose fat-soluble substances in the lipid phase and water-soluble substances in the internal aqueous phase, and can carry not only low molecular weight compounds but also high molecular weight substances such as proteins, so they are widely used as carriers for DDS of drugs, proteins, nucleic acids, etc. Research has been done. In recent years, research on practical use of liposomes provided with a targeting function or the like by binding an antibody, a sugar chain, or the like to the liposome surface in addition to the effect of reducing toxicity due to encapsulation of a drug has been progressing.

On the other hand, with respect to the general disadvantages of liposomes such as aggregation and non-specific trapping in the reticulum system such as liver and spleen, water-soluble polymers such as polyethylene glycol, polyacrylamide, polyvinylpyrrolidone, and polyglycerin are used as liposomes. It has become known that it can be improved by introduction. In particular, a great deal of research has been conducted on the modification of polyethylene glycol, and it is well known that the suppression of uptake in the reticular system by the modification of polyethylene glycol is effective.

Further, a liposome in which a targeting ligand such as an antibody is further bound to the polyethylene glycol end of a liposome modified with polyethylene glycol in order to combine the liposome with a targeting function and an effect of avoiding the retina system has been disclosed. That is, a method of forming a complex between an antibody and a liposome via a polyethylene glycol-lipid derivative having a functional group has been described, and examples thereof include the following. (Kliba
Nov et al. Liposome Res. 2 (19
92) 321, JP-A-6-126152, JP-A-6-22070, WO94 / 22429, Ha
nsen et al. BBA 1239 (1995) 133, S
hahinian et al. BBA 1237 (1995) 9
9).

In these, a lipid and a polyethylene glycol derivative are reacted in an organic solvent to synthesize a lipid-PEG-functional compound. Furthermore, after forming liposomes with other constituent lipids and binding to the antibody, antibody-PEG-
A liposome complex is obtained. Specific examples of polyethylene glycol lipid derivatives include aliphatic hydrocarbon-PEG-carbonylimidazole in JP-A-6-126152, and further JP-A-6-22070, BBA
In 1237 (1995) 99, phosphatidylethanolamine (PE) -PEG-maleimide is also used.
O94 / 22429, Hansen et al. BBA 123
9 (1995) 133, PE-PEG-NH2-Bi
otin, distearoylphosphatidylethanolamine (DSPE) -PEG-hydrazide (Hz), D
Antibody-bound liposomes using SPE-PEG-3- (2-pyridylthio) propionyl (PDP) have been disclosed.

[0010] It has been shown that these improve the antibody binding rate and improve the kinetics in blood. However, several problems were pointed out at the same time. That is, it was shown that in the method using DSPE-PEG-Hz, the antibody activity decreased during the antibody treatment, and the binding efficiency of the antibody was not significantly improved. DSPE-PEG-
In the case of PDP, a reduction treatment is required after liposome formation, which may affect the encapsulated drug, and the complicated operation is that in PE-PEG-NH2-biotin, the binding is via avidin / biotin, It is considered that the immunogenicity as a heterologous protein is considered for in vivo administration, and further, PEG is used for aliphatic hydrocarbon-PEG-carbonylimidazole.
There were problems such as the possibility that the derivative may react with the amino group of the drug to be encapsulated.

In order to bind the antibody with sufficient efficiency, it is necessary to incorporate an excess amount of the PEG lipid derivative into the liposome with respect to the antibody. Therefore, even after the antibody is bound, the PEG moiety having an active group may be incorporated into another component (in vivo administration). In the case of (1), an excess remains in a boundary region that easily reacts with the biological component). It has also been shown that the incorporation of such amounts of lipid-PEG-functional derivatives into liposomes accelerates the leakage of the liposome encapsulation (BBA).
1237 (1995) 99). The prior art was not always satisfactory in these respects.

[0012]

The inventors of the present invention have studied to solve such a conventional problem, and as a result, have found that a derivative of a synthetic water-soluble polymer, which has a reactive site with an amino group and a thiol group or a thiol group. It has been found that conventional problems can be solved by using a heterobivalent reactive water-soluble polymer having a unique thiol group (for example, S-acetylthio group) as a spacer.

[0013] Further, a heterobivalent reactive water-soluble polymer derivative is first bound to a ligand, and then bound to an active substance such as a micelle via the other end, and if necessary, reacted with the active substance. A method for easily producing a ligand conjugate using a step of bonding a monovalent water-soluble polymer having a property was found. That is, according to the present invention, a bivalent reactive water-soluble polymer derivative having a reactive site with an amino group and a thiol group portion or a latent thiol group portion; represented by the following formula (I): The above water-soluble polymer derivative;

[0014]

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(Wherein S represents a thiol group or a potential thiol group, P represents a water-soluble polymer,
N represents a reactive site with an amino group. R ¹ and R ² represent any linking group, but R ¹ and / or R ²
Is not required. A) a polymer derivative as described above, wherein the latent thiol group is an S-acetylthio group; a polymer derivative as described above, wherein the water-soluble polymer is a biodegradable polymer or polyethylene glycol; A polymer derivative; the above-mentioned polymer derivative in which a reactive site with an amino group is a succinimide group; a complex in which a ligand and an active substance are bonded via the above-described polymer derivative; represented by the following formula (II) A complex as described above;

[0016]

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(Wherein L represents an active substance, A represents a ligand, and S, R ¹ , P, R ² and N have the same meanings as described above); The above-mentioned complex selected from a molecule, a protein, a micelle and a liposome; the above-mentioned complex, wherein the active substance is a liposome or a micelle; the above-mentioned complex, wherein the ligand is an antibody;
The above-mentioned conjugate, wherein the ligand is an antibody 1-3-1; the above-mentioned conjugate, wherein the active substance is further bound to a monovalent reactive water-soluble polymer derivative having reactivity with the active substance. .

Further, a pharmaceutical composition containing the above-mentioned complex; a pharmaceutical composition as described above, which is used as an antitumor agent; a diagnostic composition containing the above-mentioned complex; Binding the water-soluble water-soluble polymer derivative to a ligand, and then bonding the water-soluble polymer derivative to the active substance via the other end of the water-soluble polymer derivative; The above-mentioned production method, comprising the step of binding a monovalent reactive water-soluble polymer derivative having the formula: wherein the bivalent reactive water-soluble polymer derivative is bound to a ligand via an amino group of the ligand. ;
The above production method, wherein the divalent reactive water-soluble polymer derivative and the active substance are bonded to the thiol group-reactive part of the active substance via the thiol group part of the divalent reactive water-soluble polymer derivative, Provided.

That is, the water-soluble polymer of the present invention (hereinafter referred to as “the polymer”)
It may also be referred to as “the present polymer compound” or “the present water-soluble polymer”. ) Is first bound to a ligand having an amino group to obtain a thiol group-added PEG-ligand complex (in the case of S-acetylthio group, after deacetylation under mild conditions), and the action of a liposome or the like provided with a thiol-reactive site. Ligand-PE easily by mixing and reacting with
A G-active substance complex could be obtained.

This eliminates the need for oxidation or reduction which may cause a decrease in the activity of a ligand such as an antibody. In addition, a complex can be formed by a reaction method via a thiol group that is unlikely to affect the active substance. Surprisingly, when an antibody and a liposome are bound via the present polymer compound, for example, when bound via a low-molecular compound having the same combination of functional groups having no polymer portion, the binding to the target cell is improved. It was found to show high binding activity. Furthermore, the effect of reducing leakage of the enclosed matter, which has been pointed out in the past, was also found.

According to this method, an excess of a functional PEG lipid derivative is introduced into a thermodynamically unstable active substance as in the conventional method in order to first form a ligand-PEG derivative such as an antibody. The formation of a ligand-PEG conjugate is possible without the need to do so. Therefore, it is possible to form an active substance by fully utilizing the conventional knowledge such as a technology for producing an active substance such as a liposome and a technique for encapsulating a drug or the like without being affected by a conventional functional group PEG lipid. It is possible.

In the conventional method, the PEG moiety having an active group remains excessively in the vicinity of the boundary where the PEG moiety having an active group easily reacts with another component (a biological component in the case of in vivo administration) even after binding of the ligand. Does not occur. In addition, by using a heterobivalent reactive water-soluble polymer derivative, there is no need to perform oxidation or reduction that may cause a decrease in the activity of a ligand such as an antibody.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The water-soluble polymer derivative of the present invention is bivalent reactive. In the present invention, bivalent reactivity refers to the case where there are two groups that can react with a functional group in the same molecule and they are different.

The water-soluble polymer used in the present invention is a synthetic polymer such as polyethylene glycol, polyacrylamide, polyvinylpyrrolidone, polyglycerin, polylactic acid, polyglycolic acid, and polyamino acid, and is preferably polyethylene glycol. Also, biodegradable polymers such as polyamino acids and polyoxy acids are preferably used.
The molecular weight is preferably about 500-20,000,
10,000 is more preferable, and 2000 is more preferable.
To 6000.

The water-soluble polymer derivative of the present invention has a thiol group or a potential thiol group at one end of the above-mentioned water-soluble polymer and a reactive site with an amino group at the other end. . A potential thiol group is a protected thiol group, which can be deprotected by appropriate treatment. Preferably, an acetylthio group is used.
The thiol group or latent thiol group may be directly added to the water-soluble polymer, or may be linked as a linking group via an alkylene, carbonyl, ester, amide, or the like.

The site reactive with an amino group refers to a site where its carboxyl group or hydroxyl group can undergo a condensation reaction with an amino group. Specifically, it can be achieved by using succinimide ester, isothiocyanate, sulfonyl chloride, carbonylimidazole or the like, and more preferably succinimide ester. In addition, the site reactive with the amino group may be directly added to the water-soluble polymer, or may be bonded via the above-described linking group.

The water-soluble polymer derivative of the present invention can be obtained by, for example, but not limited to, reacting polyethylene glycol activated with N-hydroxysuccinimide ester with mercaptoethylamine. In addition, S-acetylthioglycolic acid N-hydroxysuccinimide ester (SATA), S-acetylthiopropionic acid N-hydroxysuccinimide ester or S-acetylthioacetic acid anhydride is reacted with polyethylene glycol having an amino group and a carboxyl group to give amino acid. It can be obtained by introducing an S-acetylthio group at the terminal and further activating the carboxyl group with N-hydroxysuccinimide and N, N'-dichlorohexylcarbodiimide or p-nitrophenyl trifluoroacetate.

The bivalent reactive water-soluble polymer derivative of the present invention obtained as described above can form a complex in which a ligand and an active substance are bound. The ligand preferably has an amino group, and binds to a site reactive with the amino group of the present water-soluble polymer. Ligands include lectins, antigens, antibodies, hormones, transmitters, and the like. Preferably, it is an antibody, more preferably a 1-3-1 antibody reactive with colorectal cancer. In addition, ligands as described in the section of the description of the method for producing a complex described below can also be used.

The binding between the ligand having an amino group and the present water-soluble polymer can be easily achieved by mixing the ligand with the present water-soluble polymer in an aqueous solution and reacting at room temperature. The molar ratio of polymer derivative to ligand is about 0.3-50
Times, and more preferably about 0.8 to 30 times. In the case where a polymer having an S-acetylthio group is used as the water-soluble polymer, after binding to the ligand as described above, deacetylation is performed to form a thiol group, and then the active substance described below is used. And can be used for bonding. The deacetylation condition is not particularly limited as long as it is a mild condition in which the ligand is substantially stable. Preferably, the final concentration is about 5 mM to 200 mM in a neutral buffer solution using hydroxylamine at room temperature. Is 2
0 mM to 200 mM, more preferably 20 mM to 1
It is achieved by reacting in the range of 00 mM.

After the ligand is bound as described above, an active substance can be bound to the other end of the present water-soluble polymer. The active substance includes low molecular drugs such as methotrexate, mitomycin C, doxorubicin, and daunomycin, marker molecules such as aminofluorescein, fluorescein cadaverine, luciferyl yellow cadaverine, peroxidase, and alkaline phosphatase, neocarcinostatin, and diphtheria. Toxin A
Chain, ricin A chain, urokinase, elastase, arginase, asparaginase, superoxide dismutase, plasminogen activator, interleukin 2, proteins such as TNF, endorphin, calcitonin, bradykinin, peptide micelles such as CRF-related peptide, and liposome. However, preferred in the present invention are micelles and liposomes. In addition, pharmaceuticals and diagnostic agents may be encapsulated in the micelles and liposomes. The micelle will be described later.

Pharmaceuticals that can be encapsulated therein include antitumor agents such as adriamycin, daunomycin, vinblastine, cisplatin, mitomycin, bleomycin, actinomycin, 5-FU (fluorouracil), and the like.
Adrenergic blockers such as timolol, hypertensive agents such as clonidine, antiemetic agents such as procainamide, antimalarial agents such as chloroquinine, antibiotics such as amphotensin, and pharmaceutically acceptable salts and derivatives thereof; lysine Toxin proteins such as A and diphtheria toxin and DNAs encoding the same, DNAs encoding cytokine genes such as TNF, antisense DNA, and the like include nucleotides. As the pharmaceutically acceptable salts of the above antitumor agents and the like, salts with polyvalent anionic substances, for example, citrate, tartrate and glutamate are preferable. Particularly preferred are antitumor agents.

Further, as the diagnostic agents, radioactive elements, such as indium, imaging agents such as technetium; horseradish peroxidase, an enzyme such as alkaline phosphatase; X-ray contrast agents Yoso like;; MRI contrast agents Gadorinyumu like CO ₂ Ultrasound contrast agents, etc .; fluorescent substances, such as europium derivatives and carboxyfluorescein; and luminous substances, such as N-methylacridium derivatives. The introduction into these micelles and liposomes for pharmaceuticals and diagnostics can be carried out by a commonly used method known per se. For example, when forming micelles or liposomes, p
A method of forming a concentration gradient such as an H gradient and incorporating an ionizable drug using this potential as a driving force (C
cancel Res. 49 5922 (1989)).

The active substance is used by providing a reactive site with a thiol group by a known method. That is, a reactive group selected from a maleimide group, an alkyl halide group, an aziridine group, a pyridyldithio group, and the like can be used, but a maleimide group is preferable. Specific examples thereof include, but are not limited to, a lipid having a maleimide group such as maleimidocaproyl dipalmityl phosphatidylethanolamine (Japanese Patent Laid-Open No. 4-346918) and maleimidophenylbutyroylphosphatidylethanolamine. In the form of liposomes together with phosphatidylcholine and cholesterol according to a known method (Liposome Chapter 2 Ed. Nojima et al.
8)), a liposome having a maleimide group can be obtained. As liposomes, MLV, LU
Although various liposomes such as V and SUV can be used, LUV is preferable.

A protein having a maleimide moiety can be obtained by reacting N-succinimidyl-6-maleimidohexanoate with the protein. Further, fluorescent dyes such as maleimide fluorescein and eosine maleimide can also be used. The active substance having the thiol-reactive group is easily formed into a complex by mixing with the water-soluble polymer-ligand complex having a thiol group obtained above in a buffer near neutrality. Thus, a conjugate composed of the target ligand-PEG-active substance can be obtained. Further, if necessary, it can be purified using a technique such as ultrafiltration, gel filtration chromatography, or ion exchange chromatography, and used.

Next, the method for producing the composite of the present invention will be described in detail. FIG. 5 shows an outline of the manufacturing method of the present invention. In the figure, polymer 1 has A ′ having reactivity with functional group A in the ligand.
And a B 'moiety binding to the reactive moiety B incorporated into the active substance, ie, the above-mentioned divalent reactive water-soluble polymer derivative. Polymer 2 has B "which binds to B. B 'and B" may be the same residue. As described above, in the present invention, one substance having reactivity with an active substance is used.
A valent reactive polymer may be bound.

In the present invention, first, the heterobivalent reactive water-soluble polymer 1 is bound to the ligand, and then bound to the active substance via the other end. The method is characterized in that a complex is produced by using a step of bonding a monovalent polymer 2 having reactivity. As A in the ligand, an amino group or a sugar chain moiety can be used, but an amino group is preferable. A 'as the amino-reactive group is as described above.

The combination of B and B 'is selected from combinations in which B does not substantially react with the ligand and specifically forms a BB' bond, but a thiol group is used as B 'and a thiol reaction is used as B. Combinations of the functional groups are preferred. The thiol group may be a latent thiol group protected before the reaction as described above, that is, an S-acetylthio group. In this case, the thiol group can be used after being deprotected during the reaction.

The ligand used in the present invention is a ligand selected from proteins such as transferrin, EGF, alpha-fetoprotein (AFP), peptides such as insulin, and antibodies such as polyclonal antibody and monoclonal antibody. Even so,
It may also be a fragment obtained by enzymatic treatment or the like. When used for treatment of various diseases, mouse-human chimeric antibodies and human antibodies are preferred.

As the water-soluble polymers 1 and 2, those mentioned above as the water-soluble polymers are used, and the polymers 1 and 2 may be different combinations or the same combination. The reaction between the ligand and the present polymer 1 is as described above. As described above, when a polymer having an S-acetylthio group is used, a thiol group can be obtained by deacetylation after binding to a ligand.

In the present invention, examples of the active substance include those described above, and micelles and liposomes are particularly preferable. Therefore, hereinafter, the present invention may be described by taking micelles and liposomes as typical examples as a complex, but the active substance is not limited to micelles and liposomes. Micelles are composed of amphiphilic molecules. The amphipathic molecule constituting the micelle may be any molecule containing a hydrophilic portion and a hydrophobic portion and capable of forming a micelle by a commonly used method known per se. Suitable amphiphilic molecules include lipids.

The lipid capable of forming micelles in the present invention includes, for example, natural phosphatidylcholine such as egg yolk phosphatidylcholine (EYPC); phosphatidylcholine (PC) such as dipalmitoyl phosphatidylcholine ( DPPC), dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), and the like; natural phosphatidylethanolamine, for example, egg yolk phosphatidylethanolamine ( Phosphatidylethanolamine (PE), such as dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), etc .; phosphatidylglycero (PG), for example, dipalmitoyl phosphatidylglycerol (DPPG); phosphatidylserine (PS); phosphatidylinositol (PI); phospholipids such as phosphatidic acid (PA), glycosphingolipids, glycero Glycolipids and the like can be mentioned. These lipids are used alone or in combination of two or more, or in combination with a nonpolar substance such as cholesterol.

In the micelle of the present invention, in addition to the lipid and the non-polar substance, a lipid derivative used for binding to the ligand-polymer 1 complex and the polymer 2 is incorporated.
Examples of such a lipid derivative include a reactive site B incorporated in a micelle, that is, a phospholipid derivative having a reactive group selected from a maleimide group, an alkyl halide group, an aziridin group, and a pyridyldithio group as a thiol group reactive site. Although it can be used, it is preferably a maleimide group. Specific examples thereof include, but are not limited to, a lipid having a maleimide group such as maleimidocaproyl dipalmityl phosphatidylethanolamine (Japanese Patent Laid-Open No. 4-346918) and maleimidophenylbutyroylphosphatidylethanolamine. Can be used.

The lipid derivative is used in an amount of 0.1 to 8 mol%, preferably 0.5 to 3 mol%, based on the micelle-forming amphipathic molecule.
mol%, more preferably 1 to 3 mol%. Cholesterol is 0 to 100 mol%, preferably 40 to 100 mol% based on the micelle-forming amphipathic molecule.
It can be used at a composition ratio of 70 mol%.

The micelles in the present invention may be produced by any method, and can be produced by using the above-mentioned materials and using a commonly used production technique known per se. For example, a multilamellar liposome (MLV) formed by adding an aqueous solution to a lipid thin film adhered to a glass wall and subjecting it to mechanical shaking
And small unilamellar liposomes (S) obtained by sonication, ethanol injection, and French press
UV), surfactant removal method, reverse phase evaporation method (liposome
Junzo Sunamoto et al., Nankodo 1988), large unilamellar liposome (LUV) obtained from an extruding method or the like in which MLV is extruded by pressing a membrane having a uniform pore diameter (Liposome).
Technology, Vol. 1 2nd Edit
ion).

Further, the micelle may contain a drug or a diagnostic agent. Drugs that can be encapsulated in micelles,
The diagnostic agent is as described above. The reaction between the ligand-polymer 1 complex and the liposome or micelle is performed at about 4 ° C.
This can easily be achieved by mixing in a neutral buffer at 40 ° C. When the thiol is protected, it can be used after deprotection as described above before the reaction. P during reaction
H is preferably about 5-8, more preferably 5.5-7.
0. During the reaction, about 1 mM EDTA can be added.

The ligand-polymer 1 complex can be reacted with the reactive lipid incorporated in the micelle using not more than 2 mol equivalent, preferably not more than about mol equivalent. When the desired targeting ability can be obtained with a small amount of ligand, it is not always necessary to bind a large amount of ligand, and the reaction can be performed by adding the polymer 2 to the reactive group on the remaining micelle. When the polymer 2 is used, for example, when used as a pharmaceutical or a diagnostic agent, the micelle-polymer 1-ligand complex can be retained in blood.

In the present invention, the polymer 2 is as described above, but the polymer 2 has substantially only a covalent bond site with micelles. Although not limited, as the polymer 2, for example, a PEG derivative having a thiol group consisting of 2,4-bis (polyethylene glycol) -6-chloro-S-triazine and cysteine (JP-A-4-346)
No. 918), N-propionate-3- (thiol) methoxypolyoxyethyleneamine (J. Cont.
rolled Release 28 (1994) 15
5) can be used.

The polymer 2 may be further reacted after the reaction of the micelle with the ligand-polymer 1 complex, or may be further added after purifying the micelle-ligand-polymer 1 complex once. The addition amount is 0.2 to 5 mol equivalent of the reactive lipid incorporated in the micelle, preferably 0.5 to 2 m.
The reaction can be easily achieved by mixing at about 4 ° C. to 40 ° C. in a neutral buffer solution.

The pH at the time of the reaction is preferably about 5 to 8, more preferably 5.5 to 7.0. During the reaction, about 1 mM EDTA can be added. The ligand-bound micelle obtained in the present invention may further be subjected to ultrafiltration, if necessary,
It can be used after purification using a technique such as gel filtration chromatography or ion exchange chromatography.

[0050]

The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto without departing from the gist of the invention.

Example 1 Poly (ethylene glycol) -bis-ω-amino-α-carboxyl (PEG) dissolved in 10 ml of methylene chloride
Average molecular weight 3400, Shearwaterpolym
ers, Inc) 1 g of S-acetylthioglycolic acid N-hydroxysuccinimide ester (Sigma)
a) 74.8 mg and 41 μl of triethylamine were added. After stirring and dissolving, 10 mg of S-acetylthioglycolic acid N-hydroxysuccinimide was further added, and the mixture was stirred and reacted at room temperature for 3.5 hours. Reaction progress is TLC
(Chloroform / methanol = 85/10, iodo color development, hereinafter TLC was performed under the same conditions) and the low Rf spot of poly (ethylene glycol) -bis-ω-amino-α-carboxyl was high Rf (about 0.6).

The reaction product from which the solvent was distilled off under nitrogen was dissolved by adding 10 ml of chloroform. Sep-pak swollen with chloroform (SILICA PLUS Water
s company) and added chloroform / methanol (4/1 (v
The sample was pretreated by eluting with / v)). After evaporating the solvent again under nitrogen and dissolving in chloroform, a silica gel column (Rover column LiChroprep S) was used.
i60 25 × 310 cm (Kanto Chemical). After washing with chloroform, chloroform / methanol (85
/ 15 (V / V)) and the Rf of TLC is about 0.
Six major products were pooled and purified. The solvent was distilled off under nitrogen to obtain 583 mg. 543 mg was dissolved in about 2 ml of dehydrated methylene chloride, diethyl ether was added to precipitate, and the precipitate was collected by filtration and dried under reduced pressure with a vacuum pump. After dissolving in 5 ml of dehydrated methylene chloride, 17.8 mg of N-hydroxysuccinimide (Sigma) was added and stirred for 10 minutes. Further, 31.9 mg of N, N'-dicyclohexylcarbodiimide was added, and the mixture was reacted overnight at room temperature with stirring under a nitrogen atmosphere. After the precipitate was separated by filtration, the solvent was distilled off under nitrogen and dissolved in a small amount of dehydrated methylene chloride. Ethyl ether was added and the resulting precipitate was collected by filtration, dried under reduced pressure with a vacuum pump, and dried under TL
337 mg of a single target compound (hereinafter referred to as “Ac-S-PEG
-Suc "). 1H-NMR confirmed the production of the desired product.

[0053]

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[0054]

[Table 1]

Further, the production of the desired product was confirmed by an experiment of binding to human γ-globulin. That is, human gamma globulin (IgG, F (ab ') 2) dissolved in a buffer consisting of 50 mM phosphate buffer (pH 7.0) and 1 mM EDTA
To (4.7 mg / ml) 0.9 ml, 30 mg / ml Ac-S-PEG-Suc dissolved in dehydrated methanol was added. The addition amount was 0 to 8 times the antibody molar ratio, and the relationship between the addition amount and the amount of PEG introduced was examined. Ac-S-PEG-S
After adding uc and reacting at 25 ° C. for 1 hour, Sephacryl S1
No. 00 (Pharmacia) and purified by gel chromatography by developing with the same buffer as above, and unreacted Ac-S
-PEG-Suc (PEG molecular weight 3.4K) and antibody (molecular weight 100K) were separated.

A part of the obtained modified antibody was deprotected with hydroxylamine. That is, 0.9 ml of antibody solution
0.1 ml hydroxylamine solution (0.5 M hydroxylamine, 0.5 M HEPES, 25 mM E
DTA, pH 7.0), reacted at 25 ° C. for 10 minutes, deacetylated, and then 0.1 M phosphate buffer pH 6.0 1 mM
PD-10 equilibrated with EDTA (Pharmacia)
The low molecular weight was removed to obtain an antibody having a thiol group. The thiol group introduced into the antibody was measured according to a method using 4,4 ′ dithiopyridine (Sigma) (Seikagaku Experimental Course 5 p109, Nippon Biochemical Edition). As a control, the thiol content of each modified antibody that had not been deprotected with hydroxylamine was measured in the same manner. As a result, as shown in FIG. 1, the human γ globulin modified with Ac-S-PEG-Suc has a potential thiol group generated by deprotection with hydroxylamine, that is, an S-acetylthio group,
It was confirmed that it increased depending on the amount of Ac-S-PEG-Suc added.

Example 2 Anti-CEA mouse monoclonal antibody (IgG1F (a
b ') 2) The same procedure as in Example 1 was repeated for Ac-S-PEG-Su.
After reaction with c to obtain a modified antibody, the antibody protein was purified using a cation exchange resin. That is, the reaction solution (3.4 mg / ml
2 ml) was adjusted to pH 4 with 0.1 M acetic acid.
Added to a 2 ml bed of SP Sepharose (Pharmacia) equilibrated with 1 M acetic acid pH 4.0 buffer. After washing with 4 ml of the same buffer as above, 50 mM phosphate buffer (pH
The protein was eluted in 7.5). Centricon 30
(Amicon) The buffer was adjusted to 50 mM phosphate buffer (pH 7.0) and 1 mM EDTA with an ultrafilter, and hydroxylamine was added and deprotected in the same manner as in Example 1. The antibody to which the SH group was added was desalted with PD-10, exchanged with a 0.1 M phosphate buffer pH 6.0, and a 1 mM EDTA solution, and used for binding to a liposome described below. Ac-S-PEG-Suc and Ac-S-PEG-S
Ac-S-PEG-COO corresponding to hydrolyzate of uc
H (the compound before succinimidation in Example 1) did not bind to SP Sepharose under these conditions.

As a control, Ac-S-PEG-Suc
Is replaced by a P between the S-acetyl group and the succinimide group.
S-acetylthioglycolic acid N- having no EG moiety
Hydroxysuccinimide ester (Sigma) (hereinafter sometimes referred to as “SATA”) is reacted with the present antibody, desalted with PD-10, deprotected with hydroxylamine,
PD-10 desalting (0.1 M phosphate buffer pH 6.01
mM EDTA) to prepare an antibody having a thiol group.

The binding to the above-described antibody was examined using liposomes containing the fluorescent dye carboxyfluorescein and maleimide-modified phospholipids having a reactivity with thiol as a membrane component. That is, dipalmitoyl phosphatidylcholine (DPPC) / cholesterol (CHOL) /
Maleimidocaproyl dipalmitoyl phosphatidylethanolamine (JP-A-4-346918)
To 100 mg of the solid lipid mixture consisting of 18/10 / 0.5 (molar ratio), 1 ml of a 0.1 M carboxyfluorescein aqueous solution (adjusted to around pH 7) was added and hydrated to produce a multilamellar liposome. Further, the extruder (L
0.1 μm using IPEX Biomembranes
The resulting mixture was sized with a polycarbonate membrane of m.

The binding between the antibody and the liposome was carried out in 0.1 M citrate buffer (pH 6) at a final antibody concentration of 0.48 mg / ml and a final liposome concentration (as lipid) of 19 mg / ml.
The reaction was carried out at 5 ° C. for 1 hour. After the reaction, unreacted antibody and unencapsulated carboxyfluorescein were separated and removed by gel filtration chromatography using Sepharose CL6B (Pharmacia).

The activity of each immunoliposome thus prepared was determined by measuring the amount of fluorescence of carboxyfluorescein in which the amount of binding to a 96-well plastic plate on which an antigen was immobilized was sealed. That is, 50 μl / well of 20 ug / ml antigen CEA was added to a 96-well plate (Falcon) and fixed at 37 ° C. for 2 hours. After removing CEA, PBS
And blocked with 5% bovine serum albumin.
After washing with PBS, 50 μl / well of a liposome solution diluted to each concentration with 1% bovine serum albumin was added.
The reaction was performed for 0 minutes. After washing thoroughly with PBS, 2% trit
100 μl / well of PBS containing on × 100 and 37 ° C.
The carboxyfluorescein was eluted from the liposome by incubating for 30 minutes. A part of the solution is diluted with PBS, and the excitation wavelength is 492 nm and the fluorescence wavelength is 520 n.
m and the amount of liposomes bound to the antigen plate was compared. As a result, as shown in FIG.
The immunoliposome prepared by introducing SH into the antibody using EG-Suc showed high binding activity.

Example 3 A complex was prepared using fluorescein maleimide (Funakoshi), a maleimide-containing fluorescent dye, in place of the liposome of Example 2. That is, the SH-P shown in Embodiment 2
EG-anti-CEA antibody (0.1 M phosphate buffer pH 6.
2.5 mg / ml 0.3
5 μl of DMSO solution 3.8 mg / ml of fluorescein maleimide was added to 5 ml, and the mixture was reacted at 25 ° C. for 30 minutes with stirring.

After the reaction, unreacted fluorescein maleimide was removed by gel filtration with PD-10 (Pharmacia) equilibrated with PBS. 495nm and 280n
When the introduction ratio of fluorescein maleimide was calculated from the absorbance at m, 0.9 molecule of fluorescein maleimide was introduced per antibody. This fluorescent-labeled antibody was added to human gastric cancer cell line MKN45, and 50 μg /
The mixture was mixed for 1 hour with ice cooling under ice cooling. After washing thoroughly with PBS, binding to cancer cells was observed with a flow cytometer. As a result, as shown in FIG. 3, it was confirmed that the complex bound to cancer cells and exhibited fluorescence.

Example 4 20 mg (4 μmol) of PEG disuccinimidyl succinate (Nippon Oil & Fat Sun Bright 4001 PEG average molecular weight 5000) was dissolved in 1 ml of chloroform.
4 μmol of mercaptoethylamine hydrochloride (Sigma) dissolved in dehydrated methanol while stirring the PEG solution
Was added dropwise. Further, 56 μl of 72 mM triethylamine (in dehydrated methanol) was added. After reacting at room temperature for 7 hours under a nitrogen atmosphere, the consumption of amino group was observed by the ninhydrin reaction, and it was confirmed that the amino group was ninhydrin negative and reacted with dissuccinimide PEG. The obtained SH-PEG-succinimide was dried under a stream of nitrogen and redissolved in 1 ml of dehydrated methanol. The insolubles were filtered and used for the reaction with the following antibodies.

48.7 μl of the PEG derivative was added to 3.9 mg / ml and 0.5 ml of human γ globulin dissolved in 50 mM phosphate buffer pH 7.5 and 1 mM EDTA while stirring, and the mixture was shaken at 37 ° C. for 1 hour. Unreacted PEG
Derivative and antibody are 0.1M phosphate buffer pH 6.0 1m
Gel filtration chromatography was performed using a Sephadex G75 column (1.2 cm × 12 cm) equilibrated with M EDTA. The first eluted antibody fraction developed with the same buffer was collected and the amount of thiol introduced was quantified in the same manner as in Example 1. As a result, 0.75 mol of thiol was introduced per 1 mol of antibody. Further, the liposome was bound to the liposome in the same manner as in Example 2, and the obtained liposome was analyzed by SDS electrophoresis. As a result, the binding of the antibody was confirmed.

Example 5 Comparison of Leakage of Carboxyfluorescein A thiolated polyethylene glycol (JP-A-4-346918) was further reacted with the antibody-bound 0.1 M carboxyfluorescein-encapsulated liposome prepared in the same manner as in Example 2. An immunoliposome to which an antibody and polyethylene glycol were bound was prepared. PD equilibrated with 20 mM phosphate buffer (pH 7.5), 0.3 M NaCl
Gel filtration was performed on a -10 column to remove unencapsulated carboxyfluorescein. After further incubation at 37 ° C., the amount of carboxyfluorescein leaking from the liposomes was measured.

The leakage of carboxyfluorescein is Sh
ahinian et al. (BBA 1239 (1995) 15)
It was quantified by the method of 7). That is, after a certain period of time from the incubating liposome, the sample was sampled, diluted with the same buffer as above, and the fluorescence amount (excitation wavelength 492 nm, fluorescence wavelength 5
20 nm). At the same time, 2% SDS
And the amount of fluorescence after solubilization (broken) was measured. Since the encapsulated carboxyfluorescein is self-quenched and the fluorescence intensity reflects the amount of carboxyfluorescein leaked from the liposome, the amount of leakage was calculated based on the ratio between the two.

As shown in FIG. 4, the stability of the antibody-PEG-liposome was equal to or higher than that of the unmodified liposome (EMC-liposome). Shahinian et al. (BBA
1239 (1995) 157) is a liposome (maleimide-PEG-liposome or antibody-bound PEG-liposome) having a maleimide group introduced at the PEG tip.
Although the liposome using the EMC-PE without the EG moiety showed a larger leakage, the liposome prepared by this method showed stability equal to or higher than that of the corresponding EMC-liposome.

Example 6 In the same manner as in Example 2, carboxyfluorescein-encapsulated antibody-bound liposomes were prepared. Further, a thiolated polyethylene glycol prepared using PEG having a molecular weight of 6000 (JP-A-4-346918) was added to the liposome reaction solution. 0.1 M phosphate buffer pH 6.0 1
2.5 μmol of thiolated polyethylene glycol dissolved in mM EDTA was added to 100 mg of lipid and 1
Reacted at 0 ° C. overnight. After the reaction, Sepharose CL6B
Unreacted antibody, thiolated polyethylene glycol and non-encapsulated carboxyfluorescein were separated and removed by gel filtration chromatography using (Pharmacia).

Further, in the same manner as in Example 2, the amount of carboxyfluorescein in which the amount of binding to a 96-well plastic plate on which the antigen was immobilized was sealed was measured. As a result, in the antibody-bound liposome coated with thiolated polyethylene glycol, Ac-S-
The immunoliposome prepared by introducing SH into the antibody using PEG-Suc showed high binding activity.

Example 7 An adriamycin-encapsulated liposome (PEG immunoliposome) bound with an antibody and PEG was prepared in the same manner as in Example 5 except that adriamycin-encapsulated liposome shown below was used as the liposome and human IgG was used as the antibody. As controls, liposomes bound only with thiolated PEG (PEG liposomes) and liposomes bound neither (liposomes) were prepared.

Adriamycin-encapsulated liposomes are dipalmitoylphosphatidylcholine (DPPC) / cholesterol (CHOL) / maleimidocaproyldipalmitoylphosphatidylethanolamine 18/10 /
To 100 mg of the solid lipid mixture consisting of 0.5 (molar ratio), 1 ml of 0.3 M citrate buffer pH 4.0 was added and hydrated with a vortex mixer to produce multilamellar liposomes. Extruder (Lipex Biomembra) equipped with a polycarbonate membrane having a pore size of 1 μm.
Nes), the mixture was filtered under pressure while heating at 60 ° C. to produce sized liposomes. Furthermore, the obtained liposome solution was neutralized with 1 M NaOH, and then adriamycin (Kyowa Hakko) was added at 1/10 weight of the lipid weight to encapsulate 97% or more of adriamycin.

To compare the retention of each liposome in blood, male BALB / c mice were administered 2 mg / kg of adriamycin via the tail vein. After each hour, the animals were sacrificed and plasma was collected. The amount of adriamycin was determined by the method of Konno et al. (Cancer Res. 47 4471 (19)
87)) and extracted with hydrochloric acid ethanol, followed by quantification by fluorescence measurement.

Since free adriamycin disappears from plasma immediately after administration and is not detected under these conditions, it is considered that the detected amount of adriamycin reflects the behavior of each liposome. The results are shown in FIG. 7, and the antibody and PEG-bound liposome encapsulating adriamycin produced by this method showed high blood stability.

Example 8 The antitumor activity of immunoliposomes using the bivalent reactive water-soluble polymer derivative of the present invention in which adriamycin was encapsulated against tumor cells was verified in vitro. Using the example of a human antibody reactive to human colorectal cancer, a selective antitumor effect of the present immunoliposome on target cells was confirmed as shown below.

Colorectal cancer-reactive 1-3-1 antibody Class-switched to IgG by a conventional method and expressed in CHO cells described in JP-A-5-304987. 1 was digested with pepsin (see JP-A-4-346918).
b ′) Used as _two fragments. The above antibody fluorescently labeled with FITC was prepared for the purpose of confirming the reactivity of the antibody, and human colon cancer cell line DLD-1 (manufactured by Dainippon Pharmaceutical Co., Ltd.) and human umbilical cord vascular endothelial cell-derived SV-3T cells (Ag) were used as normal cells.
ric. Biol. Chem. 55 (11), 2847
-2853, 1991) was measured with a flow cytometer.

That is, the present fluorescent antibody was added to a BSA solution (1%
50 μg of bovine serum albumin (BSA) in PBS)
/ Ml, added to the cells, and reacted on ice for 1 hour. After washing once with BSA solution, final concentration 2μg
/ Ml propidium iodide (PI)
It was measured with a flow cytometer (FACScan Becton Dickinson). Live cells, that is, PI-negative cells were selected by a gating operation, and the value obtained by subtracting the value obtained when the antibody contained no antibody in each cell as a background value from the average channel value representing the fluorescence intensity of FITC as a background value is shown in FIG. Show. 1-3-1 antibody is SV
Higher reactivity was confirmed for DLD-1 cells than for -3T cells.

Preparation of Immunoliposome Adriamycin-encapsulated PEG immunoliposome was prepared in the same manner as in Example 7. -Cell proliferation inhibitory activity (Cytotoxicity) The present immunoliposome was serially diluted with serum derived from a healthy human to prepare a dilution series from a concentration of 40 µg / ml in terms of adriamycin. After adding to the cells and incubating at 37 ° C for 1 hour, the cells were washed once with a medium and cultured at 37 ° C. When the control without the liposome sample was confluent, the MTT assay (J. Immun
ol. Methods, 65 55 (1983)) was measured the number of cells subjected to. The cell number at each liposome concentration was expressed relative to the control cell number as 100%. FIG. 9 shows the growth inhibitory activity on human DLD-1 cells and SV-3T cells. This immunoliposome showed a more specific inhibitory effect on the target colon cancer DLD-1. That is, it is understood that the present immunoliposome has an antitumor effect.

[0079]

According to the bivalent reactive water-soluble polymer derivative of the present invention, a ligand-polymer-active substance complex can be easily formed by mixing and reacting with a ligand and an active substance such as a liposome. Obtainable. ADVANTAGE OF THE INVENTION According to the water-soluble polymer derivative of this invention, the fall of the activity of a ligand and the influence on an active substance can be prevented. According to the production method of the present invention, since a ligand such as an antibody is bound to a polymer derivative such as PEG and then bound to a micelle, an excess amount of the polymer derivative such as PEG is used relative to the thermodynamically unstable micelle. Without introduction, and therefore without these effects, ligand conjugates can be produced. Furthermore, when a drug or the like is encapsulated in the micelle in the present invention,
It is possible to reduce the leakage of the inclusion and to show high binding activity to a target molecule such as an antigen.

[Brief description of the drawings]

FIG. 1 shows the introduction of Ac-S-PEG-Suc into an antibody and the generation of a thiol group by deprotection. The horizontal axis is the antibody and Ac-
The molar ratio during the reaction of S-PEG-Suc is shown, and the vertical axis shows the number of thiol group molecules per antibody molecule generated thereby. Open circles indicate antibodies deprotected with hydroxylamine after reaction with Ac-S-PEG-Suc, and closed circles indicate antibodies that were not deprotected with hydroxylamine.

FIG. 2 shows the binding activity of a carboxyfluorescein-encapsulated liposome bound to an anti-CEA antibody into which a thiol group has been introduced using Ac-S-PEG-Suc or SATA. Each immunoliposome and the antibody-free liposome were reacted on the CEA-immobilized plate, and after washing, the amount of liposome bound to the plate was measured by fluorescence. The horizontal axis represents the amount (lipid amount) of each liposome, and the vertical axis represents the excitation wavelength of 492 n.
m, the fluorescence intensity at a fluorescence wavelength of 520 nm. Circle is A
An immunoliposome using an antibody having a thiol group introduced by c-S-PEG-Suc, a square represents an immunoliposome using an antibody having a thiol group introduced by SATA, and a cross represents a liposome without antibody binding. .

FIG. 3 shows the binding activity of a fluorescent antibody in which a thiol group-introduced antibody with Ac-S-PEG-Suc and fluorescein maleimide are bound to MKN45 cancer cells. After the reaction with the cells, the cells were analyzed using a flow cytometer. In the figure, a shows a cell group that has not reacted with the fluorescent antibody, and b shows a cell group that has reacted with the fluorescent antibody. The horizontal axis indicates the amount of fluorescence, and the vertical axis indicates the number of cells.

FIG. 4 shows a comparison of the leakage of encapsulated carboxyfluorescein from immunoliposomes (open circles) with Ac-S-PEG-Suc and the corresponding leakage from unmodified liposomes (closed squares). The vertical axis indicates the percentage of carboxyfluorescein remaining in the liposome, and the horizontal axis indicates the incubation time.

FIG. 5 shows an outline of a method for producing a ligand-bound micelle according to the present invention. A is a functional group in the ligand, A 'is a group reactive with A, and B is a reactive group introduced into micelle particles and has specific reactivity with B' and B ".

FIG. 6 shows the binding activity of a carboxyfluorescein-encapsulated liposome encapsulating thiol-containing polyethylene glycol-coated anti-CEA antibody using Ac-S-PEG-Suc or SATA, and further coated with thiolated polyethylene glycol. The horizontal axis indicates the amount of each liposome (lipid amount), and the vertical axis indicates the fluorescence intensity at an excitation wavelength of 492 nm and a fluorescence wavelength of 520 nm. Circles indicate immunoliposomes using an antibody having a thiol group introduced with Ac-S-PEG-Suc, and squares indicate
An immunoliposome using an antibody into which a thiol group has been introduced with SATA, and a cross mark indicates a liposome not bound to the antibody.

FIG. 7 shows a comparison of changes in blood concentration. The vertical axis shows the concentration of adriamycin in plasma, and the horizontal axis shows time. Each point represents the mean and SD of four animals per group. In the figure, the circle is Ac-SP
An immunoliposome using an antibody into which a thiol group has been introduced with EG-Suc represents an immunoliposome, a square represents a liposome to which only a thiolated PEG is bound, and a × represents a simple liposome to which no thiolated PEG is bound.

FIG. 8: DLD-1 cells and SV-3 of 1-3-1 antibody
It is a figure which shows the reactivity with respect to a T cell. The vertical axis indicates a value obtained by subtracting a value in the case where no antibody is contained as a background value from the average number of channels.

FIG. 9 shows a DLD of the immunoliposome obtained in Example 8.
It is a figure which shows the cell growth inhibitory activity with respect to -1 cell and SV-3T cell. The vertical axis indicates the number of cells (absorbance in the MTT assay) when the liposome sample was added, with the control being 100%. The horizontal axis shows liposomes as adriamycin concentration. □ indicates DLD-1 cells, ○
Indicates the proliferation rate of SV-3T cells.

 ──────────────────────────────────────────────────続 き Continued on front page (72) Inventor Kazuhiro Nagaike 2-5-2, Marunouchi, Chiyoda-ku, Tokyo Sanriku Chemical Co., Ltd. Pharmaceutical Company (72) Inventor Yoko Hirakawa 1000 Kamoshidacho, Aoba-ku, Yokohama-shi, Kanagawa Address: Mitsubishi Chemical Corporation Yokohama Research Laboratory (72) Inventor: Saiko Hosokawa 1000, Kamoshida-cho, Aoba-ku, Aoba-ku, Yokohama-shi, Kanagawa Prefecture Mitsubishi Yokohama Research Laboratory

Claims (20)

[Claims] 1. A bivalent reactive water-soluble polymer derivative having a reactive site with an amino group and a thiol group portion or a latent thiol group portion. 2. The water-soluble polymer derivative according to claim 1, represented by the following formula (I). Embedded image (Wherein, S represents a thiol group moiety or a potential thiol group moiety, P represents a water-soluble polymer, N represents a reactive site with an amino group, and R ¹ and R ² represent arbitrary linkages. Represents a group, but R ¹ and / or R ² are not essential.) 3. The polymer derivative according to claim 1, wherein the latent thiol group is an S-acetylthio group. 4. The polymer derivative according to claim 1, wherein the water-soluble polymer is a biodegradable polymer or polyethylene glycol. 5. The polymer derivative according to claim 1, wherein the water-soluble polymer is polyethylene glycol. 6. The polymer derivative according to claim 1, wherein the site reactive with the amino group is a succinimide group. 7. A complex in which a ligand and an active substance are bound via the polymer derivative according to any one of claims 1 to 6. 8. The complex according to claim 7, represented by the following formula (II). Embedded image (In the formula, L represents an active substance, A represents a ligand, and S, R ¹ , P, R ² and N have the same meanings as described above.) 9. The complex according to claim 7, wherein the active substance is selected from a low molecular drug, a marker molecule, a protein, a micelle and a liposome. 10. The complex according to claim 7, wherein the active substance is a liposome or a micelle. 11. The method according to claim 7, wherein the ligand is an antibody.
The complex according to any one of the above. 12. The conjugate according to claim 7, wherein the ligand is a 1-3-1 antibody. 13. The complex according to claim 7, wherein a monovalent reactive water-soluble polymer derivative reactive with the active substance is further bound to the active substance. A pharmaceutical composition comprising the complex according to any one of claims 7 to 13. 15. The pharmaceutical composition according to claim 14, which is used as an antitumor agent. A diagnostic composition comprising the complex according to any one of claims 7 to 13. 17. The bivalent reactive water-soluble polymer derivative according to claim 1, which is bound to a ligand, and then bound to an active substance via the other end of the water-soluble polymer derivative. The method for producing a composite according to any one of claims 7 to 13, comprising a step. 18. The method according to claim 17, further comprising the step of binding a monovalent reactive water-soluble polymer derivative having a reactivity with the active substance. 19. The production method according to claim 17, wherein the bivalent reactive water-soluble polymer derivative and the ligand are bound via an amino group of the ligand. 20. The divalent reactive water-soluble polymer derivative and the active substance are bonded to the thiol group-reactive part of the active substance via the thiol group of the divalent reactive water-soluble polymer derivative. The method according to claim 17.