JPH054400B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to protein-polysuccinimide complexes in which a biologically active protein is bound to a reactive polymer bound to a bifunctional ligand compound, and a method for producing the same. The protein-polysuccinimide complex according to the present invention is advantageous as an intermediate for producing a radiodiagnostic agent with a radioactive metal label in the field of nuclear medicine. That is, for example, when the physiologically active protein is fibrinogen, a diagnostic agent for a thrombus site can be produced by binding a radioactive metal to the protein-polysuccinimide complex. In addition, when the physiologically active substance is an immunoglobulin that can specifically bind to a specific antigen or a fragment thereof containing an antigen-binding site, a diagnostic agent for the purpose of detecting tumor sites, etc. can be similarly used. can be manufactured. For the purpose of detecting specific diseases, such as diagnosing the site of a blood clot, attempts have been made to label physiologically active substances made of proteins, such as fibrinogen, with radioactive substances, which have physical properties suitable for nuclear medicine diagnosis as radioactive substances. Radioactive metals are used. At that time, attempts are being made to label physiologically active substances with radioactive metals using bifunctional ligand compounds that can form chelates with the radioactive metals and form stable chemical bonds with the physiologically active substances. . However, when many bifunctional ligand compounds are reacted with a direct physiologically active substance in order to increase the specific radioactivity, the physiologically active substance is likely to be denatured. Therefore, with such conventional methods, it is difficult to obtain a nuclear medicine diagnostic agent that does not cause denaturation of physiologically active substances and has a high specific radioactivity. The present inventors conducted intensive studies to solve the above problems, and as a result, the following general formulas [1] to [6]

【formula】

【formula】

【formula】

【formula】

【formula】

[Formula] [In the above formulas [1] to [6], X, Y, W
and Z each have the following meanings. X: Reactive residue of a bifunctional ligand compound having an amino group in the molecule Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group Z: Hydrogen atom or group represented by the general formula Z' (however, , Z is a hydrogen atom, the -SH group represented by -SZ can form -S-S- bonds in equilibrium with other -SH groups within or intermolecularly; (Sh shall be expressed as SH.) Z': A group capable of forming an active disulfide bond with the adjacent sulfur atom], and each constituent unit in one molecule [1], [2], [3], [4],
The numbers [5] and [6] are n, m, l, r, respectively
p and q, 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 1,000,000, and the above problems can be solved by labeling with a radioactive metal a protein-polysuccinimide complex in which a physiologically active protein is bound to a polysuccinimide derivative [] with an average molecular weight of 2,000 to 1,000,000. The inventors have discovered that the problem can be solved, and have arrived at the present invention. The polysuccinimide derivative of the present invention [] has (1) a bifunctional ligand compound bonded to it;
It can be labeled with a radioactive metal, and (2) it has a thiol group or an active disulfide bond, so the active moiety (-SZ) and the -SH group or -NH ₂ group of a physiologically active protein can be connected. It can be used to bind to physiologically active proteins directly or via a crosslinking group. As mentioned above, the present invention is directed to a protein-polysuccinimide complex in which a reactive polymer (polysuccinimide derivative), which is important as a radiodiagnostic agent, and a physiologically active protein are bonded directly or through a crosslinking group. It provides: In the polysuccinimide derivative according to the present invention [], the "group capable of forming an active disulfide bond with the adjacent sulfur atom" represented by Z' is, for example, a 2-pyridylthio group.

[Formula] 4-pyridylthio group

[Formula] 5-carboxy-2-pyri dilthio group

[Formula] N-oxy-2- pyridylthio group

[Formula] 5-nitro-2-pyridi ruthio group

A substituted or unsubstituted pyridylthio group such as [Formula] or its N-oxide, and a 3-carboxy-4-nitrophenylthio group

[Formula] 2-nito lophenylthio group

Phenylthio groups having a nitro group such as [Formula], and 2-benzothiazoylthio group

[Formula] 2-benzimidazoylthio group

[Formula] and N-phenylamino-N'-phenyliminomethylthio group

[Formula] etc. can be cited as a specific example. The "lower alkylene group" represented by W is
For example, linear or branched alkylene groups having 1 to 4 carbon atoms such as methylene, ethylene, trimethylene, tetramethylene, propylene, and 2-methyltrimethylene can be mentioned. In the "reactive residue of a bifunctional ligand compound having an amino group in the molecule" represented by It is a general name for a compound that has a functional group that has a strong chelate-forming ability for metals and a functional group that can chemically bond two physiologically active substances. (Rediopharmaceuticals Structure−
Activity Relations, edited by Richard P. Spencer
P.282) In other words, in the present invention, a "bifunctional ligand compound having an amino group" means a compound having a chelate-forming ability for various metals, and in which the amino group in the molecule is a succinimide group of polysuccinimide.

Refers to a compound that has the ability to form an amide bond by reacting with [Formula]. In other words, it must have an amino group that reacts with a succinimide group to form an amide bond. Furthermore, in the present invention, new amino groups can be introduced not only into compounds known as bifunctional ligand compounds that themselves have amino groups, but also into bifunctional ligand compounds that do not have amino groups. These compounds are referred to as "bifunctional ligand compounds having an amino group." For example, if the bifunctional ligand compound is a compound having a carboxyl group, this carboxyl group is reacted with one amino group of a diamino compound (for example, hexamethylene diamine) to form a new amino group. can be introduced. Specific examples of "bifunctional ligand compounds having an amino group" include deferoxamine and ethylenediamine-N,N-diacetic acid (EDDA), which are known as difunctional ligand compounds containing an amino group. , 1-(p-aminoethyl)phenylpropane-1,2-dione-bis(thiosemicarbazone), etc., and as a difunctional ligand compound newly introduced with an amino group, for example, diethylenetriaminepentaacetic acid ( DTPA), ethylenediaminetriacetic acid (EDTA), etc. with amino groups introduced. Among these, deferoxamine has ⁶⁷ Ga and 1-
(p-aminoethyl)phenylpropane-1,2
-dione-bis(thiosemicarbazone) is ^99m Tc
This is preferable because it has a stable ligand ability. In particular, it is particularly desirable to use deferoxamine because the amount of labeling can be easily increased. Y is a reactive residue of a water-soluble aliphatic primary amine. Although it is not necessary to use it for the purpose of the present invention (l+r=0), it is introduced for the purpose of improving the water solubility of the reactive polymer. Examples of water-soluble aliphatic primary amines used include ethanolamine, 3
-Aliphatic primary amine having a hydroxyl group such as amino-1,2-propanediol, _CH3 ( _CH2 ) _gNH2
(g=an integer of 1 to 4), such as aliphatic amines,
In particular, amines having a hydroxyl group such as ethanolamine and 3-amino-1,2-propanediol are preferred. n+m represents the number of structural units blended with the difunctional ligand compound having an amino group, 1+r represents the number of structural units blended with the water-soluble aliphatic primary amine, and p+q represents the thiol group or the adjacent sulfur It represents the number of structural units to which groups that can form active disulfide bonds with atoms are bonded. (n+m)/(n+m+l+r+p+q) represents the number of condensed moles of X (a bifunctional ligand compound having an amino group in the molecule) per mole of succinimide group as a monomer unit. (p+q)/(n+m+l+r+p+q) represents the number of moles of the group represented by the general formula -NH-W-S-Z condensed per mole of succinimide group as a monomer unit. The arrangement state of each structural unit [1] to [6] in the polymer is not particularly limited, and may be, for example, a random copolymer or a block copolymer. The molecular weight of the polysuccinimide derivative of the present invention [] is 2,000 to 1,000,000, preferably 5,000 to 100,000, where n+m is 2 or more, (n+m)/(n+m+l
+r+p+q) is less than 1, and (p+q)/
(n+m+l+r+p+q) is 0.001 or more and 0.50 or less, preferably 0.3 or less. If the molecular weight is less than this, the number of bifunctional ligand compounds bonded to one molecule of the reactive polymer decreases, making it impossible to increase the amount of labeling with the radioactive metal, which is not preferable. Furthermore, if the molecular weight is too large, it is not preferable because the radioactive metal-labeled polymer that has been mixed in or has been cleaved in the body and is not bound to a physiologically active substance will be excreted from the body slowly. If n+m is not 2 or more, the purpose of improving the specific radioactivity of the present invention cannot be achieved. (p+q)/(n+m+l+r+p+q) is
It is 0.001 or more, and if it is less than this, the binding ratio with the physiologically active substance becomes low, which is not preferable. Also, (p+q)/(n+m+l+r+p+q) is
0.5 or less, preferably 0.3 or less; if it is too high, aggregates of physiologically active substances tend to form, resulting in insoluble matter,
This is not preferred because it tends to produce large molecular weight. The method for producing the polysuccinimide derivative of the present invention is shown in Japanese Patent Application No. 1983-91217. That is, it can be easily derived from polysuccinimide (polysuccinimide []) which is the main component of the structural unit of the following general formula []. [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. 26 , 1084 (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 dehydrated and polymerized at a temperature of 160°C or more and 240°C or less under normal pressure or reduced pressure. Polysuccinimide [ ] can be obtained by this method. The more severe the dehydration conditions are, the faster a polymer with a higher molecular weight can be obtained, but if the temperature is higher than this, the polymer will decompose, which is not preferable. If the temperature is lower than this, it will take a long time to obtain the polymer, which is not preferable. After the polymerization reaction is completed, the reaction product is immersed in DMF or
Unreacted aspartic acid and low-molecular condensate are removed by dissolving in DMSO and reprecipitating with water, followed by filtration with a glass filter and drying. Repeat this operation if necessary. Although IR and NMR suggest that the polymer thus obtained is represented by the general formula [], even if it partially contains other constituent units, it is substantially the same as the general formula [] It is generally accepted that the constituent units are the main components. It is known that the polysuccinimide thus obtained reacts with a compound having an amino group, such as ethanolamine, to carry out a reaction according to the following reaction formula. [In the formula, k, x, and y represent natural numbers. ] The polysuccinimide derivative of the present invention [] that is, a bifunctional ligand compound is bonded,
The following methods (1) to (3) can be used to obtain a reactive polymer that also has the ability to bind to a physiologically active substance. (1) Polysuccinimide [] and (a) bifunctional ligand compound having an amino group (b) general formula H ₂ N-W
A compound represented by -S-Z (W and Z are the same as above), and (c) a method for obtaining it by reaction with a water-soluble aliphatic primary amine if necessary. (2) Polysuccinimide [] and (a) bifunctional ligand compound having an amino group (b) general formula H ₂ N-W
-S-S-R (W is the same as above. R is an alkyl group (for example, a lower alkyl group having 1 to 5 carbon atoms),
It represents a hydrocarbon group such as an aralkyl group (for example, an unsubstituted or substituted phenyl alkyl group in which the alkyl group has 1 to 3 carbon atoms) or an aryl group (for example, an unsubstituted or substituted phenyl group). )
and (c) if necessary, by reacting with a water-soluble aliphatic primary amine and reductively cleaving the disulfide group of the reaction product by reacting with a thiol compound or a borohydride compound. How to get it. (3) There is a method in which the thiol-type reactive polymer (Z=H in []) obtained by any of the above methods is reacted with an active disulfide compound. Furthermore, methods (1) and (2) above include a sequential reaction in which polysuccinimide [] is reacted with the components (a), (b), and, if necessary, (c), in any order. There are reaction methods and simultaneous reaction methods in which reactions with multiple components are carried out simultaneously. Furthermore, when a reaction with the component (c) is required, a two-step method is possible in which the reaction with any one 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 for the first reaction in sequential reactions or two-step reactions, and for simultaneous reactions.
Use. The second and subsequent reactions in a sequential or two-step reaction are carried out in DMF or DMSO, or in an aqueous solvent when the polysuccinimide derivative obtained in the first or second reaction becomes water-soluble. It can be done with In the reaction methods (1) and (2) above, (a), (b), and (c)
To react the components, the following operations are performed. Polysuccinimide [] or unreacted succinimide group after the first or second reaction

Add the remaining polysuccinimide derivative of [Formula] to the solvent at a ratio of 1 to 30 (W/W).
% of one or more of the components (a), (b), and (c) to be reacted (succinimide group, which is a monomer unit of polysuccinimide [ ]) 0.001 to 10 mole per component per mole) and -40 to 80℃
Allow to react under stirring for 10 minutes to 20 days. Unreacted components can be removed by dialysis or gel filtration. Target polysuccinimide derivative []
can be obtained by distilling off the solvent under reduced pressure or by freeze-drying. Bifunctional ligand compound and general formula H ₂ N-W-
Controlling the amount of the compound represented by S-Z (W and Z are the same as above) or the compound represented by the general formula H ₂ N-W-S-S-R (W and R are the same as above). The method for doing this is to know in advance the reaction rate with the succinimide group under certain conditions when reacting with one component, and then decide the concentration of the succinimide group in the solution, temperature, reaction time, and amount of the component to be reacted. This is possible. When carrying out the reaction simultaneously with two or more components, it can be done by knowing in advance the relative reaction rate of each component in the mixture of two or more components and adjusting the mixing ratio and amount of the components to be added. Molecular weight is controlled by selecting condensation conditions to obtain polysuccinimide [ ], and polysuccinimide [ ] having the desired molecular weight is further separated by gel filtration in a DMF solution of polysuccinimide [ ]. After obtaining a fraction and a polysuccinimide derivative, molecular weight fractionation is performed by gel filtration in an aqueous solvent. After obtaining the polysuccinimide derivative [], it is preferable to perform gel filtration in an aqueous solvent to perform molecular weight fractionation. In order to improve the yield of the desired molecular weight fraction, it is necessary to select condensation conditions to obtain polysuccinimide [],
Perform molecular weight fractionation in advance by gel filtration with a DMF solution of polysuccinimide [ ]. A reactive polymer having an active disulfide group (Z=Z′ in polysuccinimide derivatives []) is obtained by reacting a thiol-type reactive polymer (Z=H in polysuccinimide derivatives []) with an active disulfide compound. The active disulfide compounds used in the method include the above-mentioned
Disulfide of the group represented by Z′, specifically
A disulfide of the above-mentioned group exemplified as Z', such as 2-pyridylsulfide, which is a disulfide of a 2-pyridylthio group.

[Formula] 4-pyridyl disulfide, which is a disulfide of 4-pyridylthio group

[Formula] 5,5'-dithiobis(2-nitrobenzoic acid), which is a disulfide of 3-carboxyl-4-nitrophenylthio group etc. 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. Alternatively, the reaction can be carried out using a reaction system in which an active disulfide compound or its acetone solution, dioxane solution, etc. are added to and mixed with an aqueous solution of the polymer. Reaction temperature: -5~70℃, reaction time: 1 minute~
24 hours is appropriate. In the protein-polysuccinimide complex that is the object of the present invention, bioactive proteins include not only bioactive proteins that have the ability to specifically bind to specific disease sites, but also partial degradation products of such bioactive proteins. In other words, it also means a fragment. Desirable physiologically active proteins in the present invention include, for example, fibrinogen, plasminogen, and urokinase, which are known to specifically bind to thrombus sites, and immune proteins that can specifically bind to specific antigens such as tumor cells. Examples include globulin and albumin, which is used as a cardiac pool diagnostic agent. In addition to diagnosing thrombus sites, fibrinogen, plasminogen, and urokinase have been used to diagnose tumor sites, taking advantage of the fact that there are many thrombus sites around tumor cells. In addition, fibrinogen is reacted with thrombin and Ca ²⁺ to form a strong fibrin clot.
Furthermore, fragment E obtained by treatment with plasmin is also known to have the ability to specifically bind to thrombus sites. As immunoglobulin, AFP, CEA,
Immunoglobulins that can specifically bind to specific antigens, such as HCG, transferrin receptor, or target cells such as tumor cells or specific lymphocytes, or tissues containing them. can be mentioned. To obtain immunoglobulin, antisera isolated from animals such as humans, monkeys, horses, cows, goats, sheep, rabbits, guinea pigs, hamsters, rats, and mice that have been immunized with antigens are fractionated with ethanol and ammonium sulfate. A method for obtaining immunoglobulin prepared by known means such as chromatography, ion exchange, or molecular sieve column chromatography, or antibody-producing cells collected from an animal immunized with an antigen, using a carcinogenic substance A method can be implemented in which hybridomas are obtained by making them cancerous or fused with myeloma cells, and monoclonal antibodies produced by these hybridomas are obtained. Immunoglobulins retain the ability to specifically bind to antigens even if their Fc portions are removed. In general, when immunoglobulin is digested with proteases 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 A so-called F(ab') ₂ fragment with two sites 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 [] and the physiologically active protein may be bound 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 (for example, a monomer obtained by reducing immunoglobulin or its fragment F(ab') ₂ 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, a reactive polymer in which Z=Z′ can be formed in a polysuccinimide derivative [ ]. This is a method of coalescence and reaction. 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, those of the general formula [a] (Here, L ₁ is a divalent organic group, and Q ₁ is an alcohol residue of imidoester.) or its salt (for example, HCl salt, HBr
salt, etc.) can be used. In the above general formula [a], the organic group represented by L ₁ is, for example, a linear or branched alkylene group having 2 to 6 carbon atoms such as ethylene or trimethylene; the alcohol residue represented by Q ₁ is Examples of the group include alkoxy groups having 1 to 3 carbon atoms such as methoxy and ethoxy.
More specifically, as the crosslinking agent [a], for example, methyl-3-mercaptopropylimidate

[Formula] Methyl-4-mercaptobutyrimidate

Examples include [Formula]. Also, 2-iminothiolactone

[Formula] N-acetyl homocysteine Antiolactone

[Formula] etc. can also be used as a crosslinking agent. Furthermore, general formula [b] [In the formula, Z′ and L ₁ are as described above.]
Q ₂ represents the alcohol residue of the active ester. ] Compound represented by general formula [c] [In the formula, Z′, L ₁ and Q ₁ are as described above. ] Compounds represented by or their salts (e.g. HCl
salt, HBr salt, etc.), general formula [d] [In the formula, L ₁ and Q ₁ are as described above. ] Compounds represented by or their salts (e.g. HCl
salt, HBr salt, etc.), general formula [c] [In the formula, L ₁ and Q ₂ are as described above. ] Compounds represented by or their salts (e.g. HCl
salt, HBr salt, etc.) and S-acetylmercaptosuccinic anhydride

[Formula] 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 [b] include N-succinimidyl-3-(2-
Pyridylthio)propionate As the crosslinking agent represented by the general formula [c], for example, 3(-4'-dithiopyridyl)mercaptopropionimidate As the crosslinking agent represented by the general formula [d], for example, dimethyl-3',3'-dithiobispropionimidate

Examples of the crosslinking agent represented by the general formula [e] include dithiobis(succinimidylpropionate)

[Formula] Disuccinimidyl (N,N'-diacetylhomocysteine) For example, The second method is to first introduce a Z' group into the mercapto group of a physiologically active protein that has a mercapto group.
A method of introducing a -SZ' group into an amino group of a physiologically active protein via a crosslinking group, and then reacting this -SZ' group with polysuccinimide [ ] where Z = hydrogen atom. It is. 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; By reacting the amino group of a physiologically active protein with a crosslinking agent represented by the above general formula [b] or [c], There is a method of directly introducing the -SZ' group. 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 This is a method in which an active protein or a physiologically active protein into which a mercapto group has been introduced and a polysuccinimide derivative [ ] in which Z = hydrogen atom are bonded using a crosslinking agent. As the crosslinking agent, a bifunctional crosslinking agent capable of reacting with a mercapto group is used. For this purpose, suitable bifunctional crosslinking agents capable of reacting with mercapto groups include, for example, the general formula []
Examples include a dimaleimide compound represented by the following formula, benzoquinone, and the like. [L ₂ represents a divalent organic group. ] As the divalent organic group represented by _L2 , for example, a phenylene group, an azobenzenediyl group, and, for example, a group of the general formula -( _CH2 ) _a1 -O-( _CH2 ) _a2-
[In the formula, a ₁ and a ₂ represent integers of 1 to 3. ]
Examples include an alkylene group via an oxygen atom represented by the following. A more specific example of a dimaleimide compound is N,N'-(1,2-phenylene) dimaleimide.

[Formula] N,N'(1,4-phenylene) dimaleimide

[Formula] 4,4'-bis(maleoyl amino)azobenzene Bis(N-maleimidomethyl)ether

[Formula] etc. can be exemplified. In order to bond a physiologically active protein and a succinimide derivative [ ] by the eighth method, a physiologically active protein having a mercapto group or a bioactive protein into which a mercapto group has been introduced is a polysuccinimide derivative [ ] in which Z = hydrogen atom. One is reacted with an excess of crosslinking agent relative to the mercapto groups it has, and then reacted 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 creates a polysuccinimide derivative in which Z=hydrogen atom [ ] This is a method of causing a reaction. Examples of functional groups that can react with mercapto groups include the formula A maleimide group represented by the general formula -CH ₂ L [wherein L is a halogen atom]. ] Examples include a halogenated methyl group represented by the following. Examples of the halogen atom include chlorine, bromine, and iodine atoms, with iodine atoms being preferred. Crosslinking agents that introduce functional groups into the amino groups of physiologically active proteins via crosslinking groups are already known;
Among them, preferred crosslinking agents include, for example, the general formula [] [In the formula, L ₃ is a divalent organic group, and Q ₂ is as described above. ] Compounds represented by the following can be mentioned. Here, as the divalent organic group, for example, a phenylene group, a general formula -( _CH2 )b-A- [wherein A is a phenylene group or a carbon number of 5 to 7
cycloalkanediyl, b represents an integer of 1 to 3; ], a group represented by the above, an alkylene group having 2 to 6 carbon atoms, and the like. Specific examples of the crosslinking agent represented by the above general formula [] include N-hydroxysuccinimide ester of m-maleimidobenzoic acid. N-hydroxysuccinimide ester of N-)4-carboxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester of N-)4-carboxyphenylmethyl)maleimide N-(ε-maleimidocaproxy)succinimide ester and so on. Examples of the crosslinking agent having a functional group represented by the general formula _-CH2L include N-hydroxysuccinimide ester of iodoacetic acid.

There is a [formula]. In methods 1, 2, 3, and 4 above, the reaction between the amino groups of the physiologically active protein and the crosslinking agent can be carried out as follows. A physiologically active protein was dissolved and prepared at a concentration of 0.5 to 100 mg/ml (preferably 1 to 20 mg/ml).
1 to 100 mol of a crosslinking agent per 1 mol of physiologically active protein is added to a buffer solution having a pH of 5 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 may be used as an aqueous solution or, if the crosslinking agent is not soluble in water, a small amount of an organic solvent (e.g. DMF, DMSO, 1,
It is used as a solution dissolved in 2-dimethoxyethane, 1,4-dioxane, methanol, ethanol, acetone, etc.). After the reaction is completed, low molecular weight substances can be removed and purified by conventional means such as dialysis or gel filtration. The reaction in the third method, in which a physiologically active protein having a mercapto group or introduced therein and a thiol-type polysuccinimide derivative 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 protein-polysuccinimide complex is performed using a physiologically active protein, a physiologically active protein into which a mercapto group has been introduced via a crosslinking group, or a modified product thereof (hereinafter, are collectively referred to as reactive physiologically active proteins), and the polysuccinimide derivatives []
and are reacted in the following manner in an appropriate combination as explained in Methods 1 to 4 above. A reactive physiologically active protein is added to a buffer solution with a pH of 5 to 9.
Its concentration is 0.5-100mg/ml (preferably 1-20mg/ml)
mg/ml) and an aqueous solution of the polysuccinimide derivative [] to be bound to the reactive physiologically active protein or the same buffer solution as above, and the mixture was heated at 0 to 50°C. Allow to react by standing or stirring gently. The polysuccinimide derivative [] is used in an amount of 0.1 to 70 moles per mole of physiologically active protein. The reaction time is usually within 3 days. Separation and purification of the target complex from the reaction mixture can be performed using common techniques such as gel filtration, affinity chromatography, dialysis, and precipitation. The complex of the present invention obtained as described above can be labeled with a radioactive metal according to a known general method. The present invention will be explained in detail below with reference to Reference Examples and Examples. Reference Example 1 Production of polysuccinimide 66.5 g of L-aspartic acid and 20.6 ml of 85% phosphoric acid
was added and heated to 190°C using a rotary evaporator, while polymerization was carried out under reduced pressure of 10 mmHg for 5.5 hours. After the polymerization reaction was completed, it was dissolved in DMF and reprecipitated with water. After re-precipitating, it was filtered through a glass filter and vacuum-dried at 80°C for 3 days to obtain polysuccinimide. When the obtained polymer was subjected to infrared absorption measurement using the KBr tablet method, absorption characteristic of succinimide groups was observed at 1800, 1720, and 1660 cm ^-1 . Molecular weight measurement was performed by GPC measurement. Using a GPC column for DMF (AD-80M/S-Showa Denko), the obtained polysuccinimide was
It was dissolved in DMF + 0.01 M LiBr solvent to a concentration of 0.2%, 250 μl of this solution was injected, and measurement was performed using a differential refractometer using the same solvent as the elution solvent. A calibration curve was created using polyethylene glycol of various molecular weights. The obtained polysuccinimide had a weight average molecular weight of 8.9×10 ⁴ and a number average molecular weight of 1.5×10 ⁴ in terms of polyethylene glycol. Reference Example 2 Method for producing immunoglobulin The method for producing a monoclonal antibody against human transferrin receptor is described by Omary MB, etal.
Described in Nature 286, 828, 1980. K562 obtained from human chronic myeloid leukemia patient
Splenic lymphocytes and non-immunoglobulin-producing myeloma cells from BALB/C mice immunized with
S194/5XXO, BU.1 strain was fused according to a conventional method, and fused cells producing antibodies against transferrin receptor were selected. The above fused cells were pre-administered with pristane.
Ascitic fluid was collected about 2 weeks after injection into BALB/C mice. Immunoglobulin was purified from ascites. 50 ascites
% saturated ammonium sulfate aqueous solution, dissolved in sodium phosphate buffer, dialyzed, obtained an IgG fraction using a DEAE-ion exchange column, and further concentrated with an Amicon membrane (PM-3 manufactured by Amicon) to give 5 mg/ml. A solution with a protein concentration of ml was obtained. Reference example 3 500mg of polysuccinimide obtained in reference example 1
(equivalent to 5.16 mmol of succinimide group) in 3.0 ml
Dissolved in DMSO and added 2530 mg (3.85 mmol) of deferoxamine mesylate (purchased from Takeda Pharmaceutical, Desferal Vial) to this solution to give 4.62 mmol.
After adding triethylamine and stirring at 60°C for 4 hours, a mixture of 16.6 mmol of cysteamine and 16.75 mmol of ethanolamine with 5 ml of DMSO was added, and the reaction was further carried out at 60°C for 1 hour. After leaving the reaction solution overnight, put it into a dialysis tube and dilute it with water.
After dialysis for 1 day, freeze-drying was performed. The polysuccinimide derivative thus obtained was dissolved in water and subjected to gel filtration using Sephadex G-50, and fractions with an average weight of 20,000 globular proteins were collected and freeze-dried. No unreacted succinimide group was detected by IR and ¹³ C-NMR measurements. The amount of deferoxamine introduced was measured by the following method. After the reaction is complete, take 400 μl of the reaction solution before dialysis and add to this.
400 μCi of ⁶⁷ Ga-citrate solution was added and allowed to stand for 5 hours to chelate ⁶⁷ Ga to deferoxamine. Thereafter, this reaction solution was separated by cellulose acetate membrane electrophoresis and radioactivity was measured. Deferoxamine introduced into the reactive polymer and unreacted deferoxamine were completely separated. The radioactivity corresponding to deferoxamine introduced into the reactive polymer was 73% of the total radioactivity. The amount of deferoxamine introduced per mole of succinimide group was determined from the following formula. [Number of moles of deferoxamine mesylate charged] x [Ratio of radioactivity introduced into the reactive polymer] / [Number of moles of succinimide groups charged] = 3.85 x 0.73 / 5.16 = 0.54 (mol) 0.54 mol per mol of succinimide group Deferoxamine has been introduced. Quantification of thiol groups was performed by elemental analysis of sulfur. The elemental analysis value of sulfur was 0.45wt%. Direct determination of thiol groups is performed using the DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] method (Anal.
Biochem, 25 , 192 (1968). i.e. polysuccinimide derivative 20mg to 8M
Dissolved in 1 ml of urea solution, 0.1 ml of 0.1 M-EDTA, 2.5%
Add 1 ml of NaBH ₄ aqueous solution and 1 ml of distilled water, add 1 drop of n-octanol, and react at 38°C for 30 minutes to reduce the disulfide bonds formed in equilibrium in the polysuccinimide derivative.
To remove NaBH ₄ , 1M − KH ₂ PO ₄ −
0.5 ml of 0.2NHCl was added, and after 5 minutes, 2 ml of acetone was added and nitrogen gas was bubbled for 5 minutes. 0.01M5,
Thiol groups were determined by adding 0.5 ml of 5'-dithiobis(2-nitrobenzoic acid) and measuring absorbance at 412 nm 15 minutes later. The quantitative results were almost in agreement with the values of elemental analysis. Molecular weight was determined by measuring the unterminated amine. 5 mg of the above intermediate in 1 ml of 0.1M sodium borate solution
Next, add 32 mg of 2,4,6-trinitrobenzenesulfonic acid (hereinafter abbreviated as TNBS) to 10 ml of
0.1ml of solution dissolved in 0.1M sodium borate solution
was added and reacted at 25℃ for 45 minutes, and then further
1.9 ml of 0.1M sodium borate solution was added, and absorption at 420 nm was measured. A molecular extinction coefficient of 11100 was obtained from a calibration curve using a known amount of aspartic acid, and the value of the unterminated amine was calculated using this molecular extinction coefficient. As a result, 1.1×10 ⁻⁴ mol of amino groups were present in 1 g, and the average molecular weight was 9,000. Reference example 4 500mg of polysuccinimide obtained in reference example 1
(equivalent to 5.16 mmol of succinimide group) in 3.0 ml
2530 mg (3.85 mmol) of deferoxamine mesylate (purchased from Takeda Pharmaceutical, Desferal Vial) was dissolved in DMSO, and 4.62 mmol of triethylamine was added. After stirring at 60°C for 4 hours, 0.67 mmol of cysteamine and ethanol were added. A mixture of 16.75 mmol of amine and 5 ml of DMSO was added, and the reaction was further carried out at 60°C for 1 hour. After leaving the reaction solution overnight, put it into a dialysis tube and dilute it with water.
After dialysis for several days, freeze-drying was performed. The polysuccinimide derivative thus obtained was dissolved in water and subjected to gel filtration using Sephadex G-50, and fractions with an average weight of 20,000 globular proteins were collected and freeze-dried. IR, ^13C -NMR
No unreacted succinimide group was detected. The amounts of deferoxamine and thiol groups introduced per mole of succinimide group were measured in the same manner as in Reference Example 3. The measurement results are 0.55 mole of deferoxamine and 0.02 mole of thiol group (amount of cysteamine introduced per mole of succinimide group).
0.02 mol). Molecular weight was determined using the same method as Reference Example 3.
It was 9000. Reference Example 5 500 mg of the polysuccinimide obtained in Reference Example 1 (equivalent to 5.16 mmol of succinimide groups) was added to 3.0 mmol of the polysuccinimide obtained in Reference Example 1.
Dissolve in DMSO and add deferoxamine mesylate (purchased from Takeda Pharmaceutical, Desferal) to this solution.
After adding 960 mg (1.47 mmol) of 3-amino-1,2- 10 mmol of propanediamine was added and the reaction was carried out at room temperature for 12 hours. After the reaction, put it in a dialysis tube and dilute it with water.
After dialysis for several days, freeze-drying was performed. 6.0 mg of the thus obtained polysuccinimide derivative
Dissolve in 0.1M Tris-HCl-1mM EDTA buffer (PH8.45) and add dithiothreitol to 0.02M in 2.0ml of the same buffer.
A solution dissolved in the solution was added, and a reduction reaction was carried out at 50°C for 100 minutes. This reaction solution was transferred to Cephadex G-50.
Perform gel filtration to obtain an average of 100,000 globular protein equivalents.
A fraction was collected and freeze-dried. When the amount of deferoxamine and thiol group introduced per mole of succinimide group were determined in the same manner as in Reference Example 3, it was found that 1 mole of succinimide group
Deferoxamine 0.25 mole to mole, 0.20
Molar thiol groups were introduced. The average molecular weight was determined to be 5000 in the same manner as in Reference Example 3. Reference Example 6 Thiol-type reactive polymer 28 obtained in Reference Example 4
mg was dissolved in 5 ml of 8M urea solution, 4.85 mg of dithiothreitol was added, and the mixture was reacted at 37°C for 1 hour. Add 5,5'-dithiobis(2-nitrobenzoic acid) to this solution.
A solution prepared by dissolving 16.07 mg in 0.15 ml of ethanol was added, and the reaction was carried out for 1 hour at room temperature. After the reaction, low molecular weight components were removed by gel filtration using Sephadex G-25, and high molecular weight components were fractionated and freeze-dried. In order to recognize that the thiol groups of the thus obtained reactive polymer having active disulfide bonds are now active disulfide bonds, it was dissolved again in 5 ml of 8M urea solution and 4.85 mg of dithiothreitol was added.
After reacting at 37°C for 1 hour, the amount of active disulfide bonds was determined by measuring absorbance at 412 nm.
As a result of the measurement, it was confirmed that almost all of the thiol groups formed active disulfide bonds. Example 1 Fibrinogen-Midori (purchased from Midori Juji) 1055 mg (fibrinogen amount 330 mg) was added to 10 ml of distilled water and stirred at room temperature for 30 minutes to dissolve. To this solution, 121 mg of dimethyl-3-3'-dithiobispropionimidate (purchased from Pierce) and 60 mg of dithiothreitol dissolved in 6 ml of distilled water were added and reacted at 23°C for 40 minutes. Low molecular components were removed by gel filtration using a 2.6 cmφ x 40 cm column of Sephadex G-25 Super Fian (manufactured by Pharmacia) equilibrated with 0.01 M phosphate buffered saline (PBS) pH 8.0. . Add 32 mg of the reactive polymer obtained in Reference Example 6 to this fractionated liquid and
The reaction was carried out at ℃ for 18 hours. After the reaction, gel filtration was performed using a 2.6 cmφ x 100 cm column of Sepharose CL-6B (manufactured by Pharmacia) equilibrated with the same buffer solution to separate the fibrinogen fraction. Protein recovery rate
Orch.
Coagulation ability was measured by the method described in Bio-chem. Biophys. 32 (1951) 317-324. An aqueous solution was adjusted so that the fibrinogen in the complex was 0.56 mg/ml, and 0.5 ml of the same buffer solution containing 50 Units/ml of Thrombin was added to 4.5 ml of this solution. After leaving it at room temperature for 1 hour, the clots ( Clot)
was removed, the fibrinogen concentration of the remaining solution was measured by absorbance measurement at 280 nm, 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 became a complex. 1 ml of an aqueous solution containing 0.56 mg/ml of the complex was added to 0.45 ml of an aqueous solution containing 670.45 mCi of gallium citrate and left for 1 hour. When the reaction solution was separated by cellulose acetate membrane electrophoresis, it was confirmed that free ⁶⁷ Ga was not present. From this, the complex is
Labeling with a specific radioactivity of 0.8 mCi/mg immunoglobulin or higher was possible, and it was confirmed that fibrinogen was bound to a reactive polymer. Example 2 A buffer solution equivalent to 31 mg of immunoglobulin obtained in Reference Example 2 was mixed with 0.1M phosphate buffer containing 1mM EDTA.
The buffer solution was exchanged by applying it to a Sephadex G-25 (manufactured by Pharmacia) column equilibrated with PH7.0 (hereinafter referred to as buffer A), and concentrated to 5 mg/ml using an Amicon membrane (manufactured by Amicon - PM-10). liquid 6
N-)4-carboxycyclohexylmethyl) in ml
Add 30μl of a 40mM dioxane solution of maleimide N-hydroxysuccinimide ester at 30°C.
The reaction was carried out for 1 hour. After the reaction, low molecular weight components were removed by gel filtration using Buffer A using a 2.0 cmφ x 20 cm column of Cephadex G-25 Superfine (manufactured by Pharmacia). 30 mg of the reactive polymer obtained in Reference Example 4 was added to the buffer solution.
Dissolve dithiothreitol 13 in this solution in 3 ml of A
After reaction at 30°C for 2 hours, gel filtration was performed in the same manner as above to remove low molecular weight 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, 30ml of the reaction solution was applied to a column equilibrated with 0.1M phosphate buffer (PH8.20) using Protein A (manufactured by Pharmacia), and the non-adsorbed components were removed with the equilibration buffer. After thorough removal, the components adsorbed on the protein A column were eluted with a 0.58% acetic acid-0.15N NaCl solution. The eluate was dialyzed against 1 liter of 0.1M carbonate/bicarbonate buffer (PH9.3). After dialysis, the dialysate was concentrated using an Amicon membrane (PM-10 manufactured by Amicon) to obtain 6 ml of a solution with a protein concentration of 1.18 mg/ml. In order to confirm that the thus obtained complex was a combination of immunoglobulin and a reactive polymer, the concentrated solution was used and labeled with ⁶⁷ Ga in the same manner as in Example 1.
0.90 ml of a solution containing 0.90 mCi of gallium citrate was added to 1 ml of the concentrate and left for 1 hour. When the resulting reaction solution was separated by cellulose acetate membrane electrophoresis, it was confirmed that free gallium was not present. This confirmed that the complex could be labeled with a specific radioactivity of 0.8 mCi/mg immunoglobulin or higher, and that the immunoglobulin and the reactive polymer were bound together. In order to confirm that the immunoglobulin complex retains the antigen binding ability as an antibody, the following experiment was conducted. Immunoglobulin was iodine-labeled by the chloramine-T method. 0.5M in plastic tube
Add 5 μl of sodium phosphate buffer PH7.5 (hereinafter referred to as buffer B) and 0.3 mCi of Na ¹²⁵ I, stir, and then add 200 μg/ml chloramine-T in buffer B solution.
After adding 20 μl and stirring for 20 seconds, 20 μl of a 0.5 mg/ml immunoglobulin buffer B solution was added and the reaction was carried out with stirring for 15 seconds. After the reaction, 400 μl of 14 μg/ml Na ₂ S ₂ O ₅ in buffer B solution, 25 μl of 20 mg/ml KI in buffer B solution,
25 μl of 5% BSA in Buffer B was added. Thereafter, gel filtration was performed using a Sephadex G-25 column using buffer B as an eluent, and the protein eluate was fractionated to remove low molecules. The thus obtained ¹²⁵ I-
To determine the specific radioactivity of labeled immunoglobulin,
The protein concentration was determined from the absorption at 280 nm, and the total radioactivity was measured, and the specific radioactivity was 4.3 mCi/mg. 1 ^{ml of} 10% FCS-
In addition to RPM1-1640 medium (purchased from Hani Chemical), add 1.0 to 0.1 mg/ml of the above complex to 0.1 ml of this solution.
Add 0.4 ml of 10% FCS-RPM1-1640 medium solution dissolved so that
10% FCS of CCRF-CEM cells at 1×10 ⁶ cells/ml
-Add 0.5ml of RPM1-1640 medium solution and 4℃
Incubation was carried out for 1 hour. After this
Centrifuge at 2000rpm for 5 minutes and discard the supernatant.
After adding 0.5% BSA-Hank's buffer to the sediment
Centrifuge at 2000rpm for 5 minutes and discard the supernatant.
The radioactivity of the sediment was measured. For comparison, we conducted an experiment by adding immunoglobulin at the same concentration instead of the complex.
The complex and immunoglobulin have the same effect of inhibiting the binding of ¹²⁵ I-labeled immunoglobulin to CCRM-CEM cells, indicating that the complex immunoglobulin has the same binding ability as the original immunoglobulin. Shown. Example 3 The immunoglobulin obtained in Reference Example 2 was used in Example 2.
Similarly, exchange to 0.1M phosphate buffer (buffer A) containing 1mMEDTA to make 6ml of 5.5mg/ml solution.
30 μl of an ethanol solution of 20 mM N-succinimidyl-3-(2-pyridylthio)propionate was added and incubated at 23° C. for 30 minutes. After the reaction, 5 mg of glycine as a reaction terminator and 5 mg of dithiothreitol as a reducing agent were added and incubated at 23°C for 30 minutes. After that, 2.0 cmφ of Cephadex G-25 (manufactured by Pharmacia) was equilibrated with buffer A.
Gel filtration was performed using a x20 cm column to remove low molecular weight components, and 7 ml of protein fraction was collected. To this aliquot, 10 mg of a reactive polymer having active disulfide bonds obtained in the same manner as in Reference Example 6 was added, and the mixture was incubated at 4°C for 18 hours. Example 2 for the purpose of removing the unreacted reactive polymer after that.
After separation using a protein A column in the same manner as above, concentration was performed to obtain approximately 6 ml of a solution with a protein concentration of 1.12 mg/ml. When labeling and separation with gallium-67 was carried out under the same conditions as in Example 2, free gallium was not observed, and a complex with a specific radioactivity of 0.8 mCi/mg of immunoglobulin or more was formed, indicating that it was not an immunoglobulin. It was confirmed that the and reactive polymer were bonded together. Confirmation of whether the immunoglobulin in the complex retains the ability to bind to the antigen as an antibody is performed in the same manner as in Example ² . When a comparative method was used, it was confirmed that the binding ability did not decrease. Example 4 1.2g of immunoglobulin was added to 0.1M acetate buffer (PH
4.5) After dissolving in 40 ml and adding 24 mg of -pepsin to decompose at 37°C for about 18 hours, the decomposed product was applied to a 3.5 cmφ x 140 cm column of Cephadex G-200 (manufactured by Pharmacia) in physiological saline. , extract the protein that flows out at a molecular weight of approximately 100,000 F
(ab′) ₂ was obtained. _10mM phosphate buffer (PH
6.5) N,N containing 0.42 mg of N-hydroxysuccinimidyl-m-maleimidobenzoic acid in 1.14 ml
- Add 0.05 ml of dimethylformamide and at room temperature
The reaction was allowed to proceed for 40 minutes. After the reaction, 5mM acetate buffer containing 0.14M sodium chloride and 1mM-EDTA PH5.5
Excess reagent was removed by dialysis to obtain m-maleimidobenzoyl F(ab') ₂ . 41mg of the reactive polymer obtained in Reference Example 4 was added to 0.1M
Sodium phosphate-1mMEDTA buffer (PH7.0)
Dissolve 13mg of dithiothreitol in 3ml of this solution.
After reaction at 30℃ for 2 hours, add 5mM acetate buffer PH containing 0.14M sodium chloride and 1mM-EDTA.
2.0 cm x 13.5 cm of Cephadex G-25 Super Fine (manufactured by Pharmacia) equilibrated at 5.5
Gel filtration was performed using a cm column to remove low molecular components. Mix a buffer equivalent to 30 mg of m-maleimidobenzoyl F(ab') ₂ and a buffer equivalent to 15 mg of reactive polymer, add 0.3 ml of 0.5 M phosphate buffer (PH6.5), and mix at 4°C.
It reacted for 16 hours. After the reaction, it was dialyzed against 0.9% sodium chloride solution. The complex obtained as above is treated with immunoglobulin.
Concentrate to a concentration of 1.1 mg/ml, and ^reduce to 1 ml after concentration.6
⁷ 4 mCi of Ga-citric acid solution was added and left for 1 hour.
When the resulting reaction solution was separated by cellulose acetate membrane electrophoresis, it was confirmed that free gallium was not present. Specific binding of the complex labeled with ⁶⁷ Ga to CCRF-CEM cells containing human transferrin receptor was confirmed. CCRF-CEM cells or FM without human transferrin receptors-
3A cells 10 ⁶ Cells/ml solution at each dilution ratio 0.5
Add 5×10 ⁴ cpm of ⁶⁷ Ga-labeled complex to 1 ml of
Add 0.4 ml of a solution dissolved in 10% FCS-RPM1-1640 medium (purchased from Hani Chemical), incubate at 4°C for 1 hour, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, and add 0.5% BSA. −
After adding 0.5 ml of Hank's solution (purchased from Hani Chemical), washing and centrifuging at 2000 rpm for 5 minutes, the supernatant was discarded and the radioactivity of the precipitate was measured using a γ-counter. The results are shown in the table below.

[Table] The above results confirmed that the complex was indeed synthesized and that the ability of F(ab') ₂ to bind to the antigen was maintained. Example 5 Immunoglobulin was digested with pepsin in the same manner as in Example 4 to obtain (Fab') ₂ . 0.1M Sodium Phosphate containing 40mg of F(ab') ₂ - 1mM MEDTA (PH6.0)
Buffer solution (hereinafter referred to as buffer solution C) 3.43ml, 0.1M2
-1 ml of buffer C solution of mercaptoethylamine
In addition, reduction was performed at 37°C for 90 minutes. After the reaction, the solution was equilibrated with buffer C using Sephadex G-25.
(manufactured by Pharmacia) to remove low molecular weight substances. After separating the protein fraction, N,N'-O-
0.1M acetic acid of phenylene dimaleimide
25.7ml of saturated solution to 1mM MEDTA buffer PH5.0
was added and reacted for 20 minutes at 30°C with stirring. 0.02M after reaction
Sephadex equilibrated with acetate buffer PH5.0
Low molecular weight components were removed using G-25, followed by concentration to obtain a solution of Fab' having a maleimide group introduced therein at a concentration of 2.5 mg/ml. 41mg of the reactive polymer obtained in Reference Example 4 was added to 0.1M
Sodium phosphate-1mMEDTA buffer (PH7.0)
Dissolve 13mg of dithiothreitol in 3ml of this solution.
After reaction at 30°C for 2 hours, low molecular weight components were removed by gel filtration using Sephadex G-25 Superfine equilibrated with 5mM acetate buffer pH 5.5 containing 0.14M sodium chloride-1mM EDTA. Mix a buffer solution equivalent to 15 mg of Fab′ into which a maleimide group has been introduced and a buffer solution equivalent to 15 mg of reactive polymer, and then
Add 0.3ml of 0.5M phosphate buffer (PH6.5) and heat at 4℃ for 16 minutes.
Time reacted. After the reaction, it was dialyzed against 0.9% sodium chloride solution. After labeling the obtained complex with ⁶⁷ Ga in the same manner as in Example 4, the binding of the complex to CCRF-CEM cells and FM-3A cells was examined, and the following data were obtained.

[Table] From the above results, it was confirmed that the complex was indeed synthesized and that the binding ability of Fab' to the antigen was maintained. Test Example: Behavior of protein-polysuccinimide complex in rats after labeling with gallium-67 5 mg of the protein-polysuccinimide complex produced by the method of Example 2 was injected with gallium-67 citrate.
A gallium-67 labeled reactive polymer was obtained by adding to 2 ml of a solution containing 2 mCi and leaving it for 1 hour. The absence of free gallium-67 was confirmed by cellulose acetate membrane electrophoresis. This gallium
0.2 ml of the 67-labeled reactive polymer solution was intravenously administered to three female SD rats. One hour after administration, each organ of the rats was removed and the uptake rate in each organ was determined by measuring radioactivity. The results are as follows.

[Table] As described above, the labeled substance was present in the blood and there was little accumulation in other organs, indicating that the complex was useful as a nuclear medicine diagnostic agent.

Claims (1)

[Claims] 1 The following general formulas [1] to [6] [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] [In the above formulas [1] to [6], X, Y,W
and Z each have the following meanings. X: Reactive residue of a bifunctional ligand compound having an amino group in the molecule Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group Z: Hydrogen atom or group represented by the general formula Z' (however, , Z is a hydrogen atom, the -SH group represented by -SZ can form -S-S- bonds in equilibrium with other -SH groups within or intermolecularly; (Sh shall be expressed as SH.) Z': A group capable of forming an active disulfide bond with the adjacent sulfur atom], and each constituent unit in one molecule [1], [2], [3], [4],
The numbers of [5] and [6] are n, m, 1, r, respectively
p and q, these numbers are 0 or natural numbers, and the relationship between them is n+m≧2 0.001≦(p+q)/(n+m+1+r+p+
q) A protein-polysuccinimide complex in which a physiologically active protein is bound to a polysuccinimide derivative [] that satisfies ≦0.50 and has an average molecular weight of 2,000 to 1,000,000. 2. A patent in which the bond between the polysuccinimide derivative [] and the physiologically active protein is a disulfide bond formed by using the -SZ group of the polysuccinimide derivative [] and the mercapto group of the physiologically active protein having a mercapto group. The protein-polysuccinimide complex according to claim 1. 3. A patent claim in which the bond between the polysuccinimide derivative [] and the physiologically active protein is a bond that utilizes the -SZ group of the polysuccinimide derivative [] and the amino group, which has little bioactivity, and that a crosslinking group is interposed between them. The protein-polysuccinimide complex according to item 1. 4 The bond between the polysuccinimide derivative [] and the physiologically active protein utilizes the -SZ group of the polysuccinimide derivative [] and the mercapto group of the physiologically active protein having a mercapto group, and a crosslinking group is created between them. 2. The protein-polysuccinimide complex according to claim 1, wherein the protein-polysuccinimide complex is bonded through . 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] [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] [In the above formulas [1] to [6], X, Y, W
and Z each have the following meanings. X: Reactive residue of a bifunctional ligand compound having an amino group in the molecule Y: Water-soluble aliphatic primary amine residue W: Lower alkylene group Z: Hydrogen atom or group represented by the general formula Z' (however, , Z is a hydrogen atom, the -SH group represented by -SZ can form -S-S- bonds in equilibrium with other -SH groups within or intermolecularly; (Sh shall be expressed as SH.) Z': A group capable of forming an active disulfide bond with the adjacent sulfur atom], and each constituent unit in one molecule [1], [2], [3], [4],
The numbers [5] and [6] are n, m, l, r, respectively
p and q, 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 1,000,000.In producing a protein-polysuccinimide complex in which a physiologically active protein is bound to a polysuccinimide derivative [] with an average molecular weight of 2,000 to 1,000,000, The above-mentioned protein-polysuccinimide complex, characterized in that the polysuccinimide derivative [ ] and the physiologically active protein are bonded by using a mercapto group newly introduced into the active protein and a mercapto group of the polysuccinimide derivative [ ] How the body is made. 7 A method of bonding using a mercapto group of a physiologically active protein and a polysuccinimide derivative [] can be used to bond a physiologically active protein with a mercapto group, or a physiologically active protein into which a mercapto group has been introduced via a crosslinking group of an amino group, 7. The method for producing a protein-polysuccinimide complex according to claim 6, which is a method of reacting with a polysuccinimide derivative [] where Z=Z'. 8 A method of bonding using the mercapto group of a physiologically active protein and a polysuccinimide derivative [] involves introducing a Z' group into the mercapto group of a physiologically active protein having a mercapto group to form a -SZ' group, or via a cross-linking group to the amino group of a physiologically active protein.
A bioactive protein having an SZ' group introduced in this manner or having an -SZ' group introduced through a crosslinking group, and a polysuccinimide derivative in which Z=hydrogen atom The method for producing a protein-polysuccinimide complex according to claim 6, which is a method of reacting with [ ]. 9 A method of bonding using a mercapto group of a physiologically active protein and a polysuccinimide derivative [] can be used to bond a physiologically active protein with a mercapto group, or a physiologically active protein with a mercapto group introduced into an amino group via a crosslinking group, It has a mercapto group (Z=
A method according to claim 6, which is a method in which one of the polysuccinimide derivatives (which is a hydrogen atom) is reacted with an excess of a crosslinking agent with respect to the mercapto group it has, and then the other is reacted with the other polysuccinimide derivative. Method for producing protein-polysuccinimide complex. 10 The method of bonding using the mercapto group of a physiologically active protein and a polysuccinimide derivative [] produces a physiologically 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 a polysuccinimide derivative in which Z=hydrogen atom.