JPH0428000B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing protein or polypeptide conjugates of bleomycin. Bleomycin in 1966
An anticancer antibiotic discovered by Umezawa et al., one of the inventors of the invention, in 2010 (Umezawa et al.: Journal of
Antibiotics, p. 200, 1966) 1 produced by the actinomycete Streptomyces verticillus.
It is a basic water-soluble glycopeptide that easily chelates divalent copper atoms, and 16 types of copper-containing bodies have been produced and isolated using conventional culture methods. (for example,
Umezawa et al.: Journal of Antibiotics
19A, p. 210 1966). Among these bleomycins, A1, A2, A5, B2, demethyl A2, etc. are already widely used in the clinical field of cancer treatment in the copper-free form of their mixture (hereinafter referred to as "bleomycin complex"). In particular, it has achieved excellent results in treating squamous cell carcinoma, skin cancer, head and neck cancer, lung cancer, and malignant lymphoma. Also, U.S. Patent No. 3922262 and U.S. Patent No.
Re30451 discloses various bleomycins. Due to the need for radioimmunoassay (hereinafter referred to as RIA) and enzyme immunoassay (hereinafter referred to as EIA) for bleomycins and their derivatives, the present inventors
We investigated methods of binding bleomycin to proteins or polypeptides. Three previous examples of binding bleomycins to proteins have been found in the literature. A.Brouton & J.E.
Strong reacted decoppered bleomycin complex with bovine serum albumin (BSA) using water-soluble carbodiimide in a phosphate buffer to obtain a bleomycin-BSA conjugate, which was used for RIA of bleomycin ( Cancer
Research 36 1419-1421, 1976). Also K.
FuJiwara, M. Yasuno, K. Kitagawa et al. GMBS
(N-γ-(maleimido-butyloxy)-
acylate pepromycin with succinimide),
A maleimide group was introduced and then an SH group was introduced.
A peplomycin-BSA conjugate is synthesized by reacting with BSA. At the same time, MBS (N-m-
A maleimide group is introduced by acylating copperless pepromycin with (maleimidobenzoyloxy succinimide), and this is reacted with the SH group of β-galactosidase to synthesize a pepromycin-β-galactosidase conjugate. (Canser Research 41 4121-4126, 1981) However, it has not been reported that bleomycin (or its derivatives)-protein conjugates made by any of the above methods have the biological activity of bleomycins, and Acylate the copper body or
Since the conjugate is exposed to reaction conditions that generate an amide bond, the primary amino group of the diaminopropionic acid amide moiety, which is essential for the expression of bleomycin activity, is used in the bonding reaction and the biological activity of bleomycin is lost. It is thought that it is on. In addition, in the conjugate obtained by the above binding method, the terminal amine moiety of bleomycin and pepromycin is free, so the antiserum made using these also recognizes the terminal amine of bleomycin and pepromycin, and it is difficult to distinguish between the two. Conceivable. Therefore, in the above method, it is considered necessary to prepare separate antisera for each of bleomycin and pepromycin. Therefore, the present inventors investigated various methods for producing bleomycin protein or peptide conjugates that retain the activity of bleomycin and can produce antiserum that recognizes various bleomycins. After protecting the active site of the bleomycin derivative as a copper complex, the bleomycin obtained by introducing a group having a carboxylic acid or a reactive derivative thereof into the terminal amino group is condensed with a protein or peptide to form a bleomycin-protein or peptide. It has been found that a conjugate can be obtained. That is, the present invention is based on the following general formula [] [BX]-NH-A-NR _2-o (CH ₂ Y) _o [] [where [BX] is the following formula It can be expressed as (in the case of copper-containing substances, chelate copper is omitted)
Represents a residue obtained by removing the hydroxyl group from the carboxyl group of bleomycin acid, A represents a bonding chain connecting an amino group, and lower alkylene or alkylene bonded via an N atom, such as -(CH ₂ ) _n -NCH ₃ - (CH ₂ ) _n ′−, −(CH ₂ ) _n −
NH−(CH ₂ ) _n ′−,

[Formula] (m and m' are integers of 2 to 6, preferably 3 or 4), R is hydrogen, or an alkyl group,
or an alkyl group substituted with phenyl, e.g.
_CH3 ,

[Formula], n is 1 or 2, and Y represents a carboxyl group or a reactive derivative thereof. The present invention relates to the production of a protein or polypeptide conjugate of bleomycins, which is characterized by binding a protein or polypeptide to a novel bleomycin derivative represented by the following. In the protein or polypeptide conjugate of bleomycin obtained in the present invention, the number of bleomycins bound to one protein molecule varies depending on the reaction method or the type of protein, but generally 1 to several tens of bleomycins are bound to one protein molecule. is within the range of The bleomycin derivative of the general formula [] used in the present invention can be used in either a copper-containing form or a copper-free form. For the condensation of the bleomycin derivative of the above general formula [] with a protein or polypeptide, a method normally used for peptide condensation can be used. For example, when Y is a carboxyl group, a commonly used condensing agent can be used. The protein or polypeptide may be condensed in the presence of Y, or if Y is a reactive derivative, it may be condensed with the protein or polypeptide as is, with the addition of a condensing agent if necessary. Among the compounds represented by the above general formula [],
A is preferable because it is easy to manufacture.
is −(CH ₂ ) ₃ −, −(CH ₂ ) ₃ −NCH ₃ −(CH ₂ ) ₃ −, −
(CH ₂ ) ₃ −NH− (CH ₂ ) ₃ −, −(CH ₂ ) ₃ −NH−
Examples include compounds in which ( _CH2 ) _4- ,R is hydrogen or 1-phenylethyl. The reactive derivative of the carboxyl group represented by Y in the above general formula [] is not particularly limited as long as it can react with the amino group of a protein or polypeptide, but active esters that are usually used to form peptide bonds Derivatives are used. Representative compounds of the general formula [] include the compounds shown in Table 1. In addition, in the table, X represents a group obtained by removing carbonyl from Y.

[Table] The compound represented by the above general formula [] can be produced as follows. General formula [] [BX]-NH-A-NH-R [] [In the formula, [BX], A, and R are the same as above. ] By reductively condensing OHC-COOH or a salt thereof as a carbonyl compound to the bleomycin derivative represented by
It is possible to obtain a compound in which is a carboxyl group. Examples of the reducing agent used in the condensation include borohydride compounds such as sodium cyanoborohydride. Catalytic reduction may also be carried out using a catalyst such as palladium on carbon. When R is H in the carbonyl compound, if 1 to 1.5 mol is used, a derivative with n=1 is mainly obtained, and when 3 mol or more is used, a derivative with n=2 is mainly obtained. When R is other than H, it may be used in an amount of 1 mole or more. The reaction uses methanol, water, dimethylformamide, acetonitrile, and a mixture thereof as a solvent. Additionally, salts such as sodium acetate, potassium acetate, and sodium phosphate, and acetic acid may be used alone or in a mixture at an appropriate ratio. The temperature is preferably 0 to 50°C. To isolate the derivative obtained as above, when using a borohydride compound, adjust the pH of the reaction solution to 1 with hydrochloric acid, stir at room temperature for 5 to 10 minutes, and remove excess reducing agent. After decomposition and neutralization, methanol was distilled off under reduced pressure, and excess aldehyde and ketone were extracted and removed with ether or butanol, followed by the next desalting operation. That is, an adsorption resin such as Amberlite XAD-2 (manufactured by Rohm and Haas) is injected into a column filled with distilled water to adsorb the target substance. After washing away the salts with distilled water, add acidic water-containing methanol, e.g. 1/50
Elute with a normal aqueous hydrochloric acid solution-methanol (1:4 v/v), collect the blue bleomycin derivative fraction, and if necessary, use an anion exchange resin or Dowex.
44 (OH type; manufactured by The Dow Chemical Company), concentrated under reduced pressure, and freeze-dried to obtain a blue coarse powder of the derivative. To further increase the purity, perform the following operation. Dissolve the above powder in distilled water and adjust the pH beforehand.
CM Sephadex C-25 (Na+
The sample is injected into a column filled with Pharmacia (manufactured by Fine Chemical Co.) and adsorbed. Elution is performed by a linear concentration distribution method in which the sodium concentration is gradually increased to 1.0M by continuously adding sodium chloride to the above buffer solution. When Y is a carboxyl group, in this chromatography, the derivative with n=2 elutes fastest, followed by the derivative with n=1, and the unreacted raw material elutes last, so an ultraviolet absorption monitor is used. It is possible to separate by using . If impurities are found in the fraction of the target product, either repeat the above chromatography or use a PH6.8 buffer instead of the PH6.8 buffer.
Complete removal can be achieved by repeating chromatography using 4.5 1/20 molar acetic acid-sodium acetate buffer. The thus obtained fraction of the target product was desalted using the Amberlite XAD-2 desalting method used previously, and then freeze-dried to obtain the copper-containing substance of the bleomycin derivative as a blue amorphous powder. It will be done. When the bleomycin derivative produced by the method described above is hydrolyzed in 6N hydrochloric acid at 105°C for 20 hours, decomposition products common to BLMs [L-threonine, β-amino-β-(4
-amino-6-carboxy-5-methyl-pyrimidin-2-yl)propionic acid, 4-amino-3
-oxy-2-methyl-n-pentanoic acid, β-oxy-L-histidine, β-amino-L-alanine, 2'-(2-aminoethyl)-2,4'-bithiazole-4-carboxylic acid] and an amine represented by the general formula [] containing a carboxyl group corresponding to the raw material bleomycin of the general formula [] NH ₂ -A-NR _2-o (CH ₂ COOH) _o [[]] [wherein A and R are the same as above] . ] was detected. Furthermore, in methanolysis using Amberlyst 15, the same L-gulose and 3-0-carbamoyl-D-mannose methyl glycosides as bleomycin were detected by gas chromatography. The above facts support that the bleomycin derivative produced by the method of the present invention has the chemical structure represented by the above formula []. Further, a compound in which Y is a reactive derivative of a carboxyl group in the general formula [] can be produced, for example, as follows. It can be produced by condensing a compound represented by the general formula [] in which Y is a carboxyl group with a compound represented by the general formula [] HX [] [wherein X is the same as above] using a condensing agent. As the condensing agent, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-cyclohexyl-
3-(2-morpholinoethyl)carbodiimide,
Examples include diisopropylcarbodiimide, diphenylphosphorazidate (DPPA), and diethylphosphorocyanidate (DEPC). As a compound represented by the general formula [], p-
Nitrophenol, o, p-dinitrophenol, pentachlorophenol, 2.4.5-trichlorophenol, pentafluorophenol, N-
Hydroxysuccinimide, N-hydroxy-5
-Norbornene-2,3-dicarboximide and the like. The solvent used in this condensation may be any solvent as long as it does not affect the reaction, but polar solvents that dissolve the raw material compounds represented by the general formulas [] and [] are usually preferred, such as water, dimethylformamide,
Dimethylacetamide, acetonitrile, and a mixed solvent thereof. The ratio of the compound of general formula [] and the condensing agent to the compound of general formula [] is
It is 0.5 to 20 equivalents, preferably 1 to 10 equivalents. The condensate obtained in this way is the desired active ester, as shown by the IR absorption spectrum of 1780,
This is clear from the fact that it shows an absorption band around 1740 cm ^-1 and then forms a bond with protein, and that it easily returns to its original compound with a carboxyl group when hydrolyzed with cold alkaline water. When Y is a reactive derivative of a carboxyl group, the compound of the general formula [] thus obtained can be prepared by simply reacting with a protein or polypeptide in a neutral or slightly alkaline buffer.
In the case of a compound in which Y is a carboxyl group, the protein or polypeptide is treated with a commonly used condensing agent, such as the above-mentioned condensing agent, preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. A bleomycin-protein or polypeptide conjugate can be obtained by the reaction. The reaction temperature is usually -5° to 60°C, preferably 0°C to 30°C. In the present invention, the ratio of the protein or polypeptide to the compound of the general formula [] is not particularly limited, but it is usually preferable to use the compound of the general formula [] in excess of the protein or polypeptide. By increasing the amount of the compound, the amount of bleomycin bound to the protein or polypeptide can be increased. The above proteins or polypeptides include albumin, globulin, enzyme,
and polylysine. To isolate the target bleomycin-protein or polypeptide conjugate from the above reaction solution, methods commonly used for protein or polypeptide purification are used, such as gel filtration, ultrafiltration, salting out, and dialysis. are all used. To give an example, the above active ester (copper-containing body)
The bleomycin-BSA conjugate prepared using
When injected into a Sephadex G50 column equilibrated with a phosphate buffer and subjected to chromatography, it separates into two blue fractions. The blue substance with a large molecular weight that is eluted first is the target substance (copper-containing substance), and the blue fraction that is eluted later contains the active ester of the raw material and its hydrolyzate. When the desired fraction is dialyzed against water and then freeze-dried, a bleomycin-BSA conjugate (copper-containing compound) is obtained as a blue powder. This product is useful as an antigen for immunization to create antiserum for use in EIA and RIA. Product structure Gel chromatography shows that the molecular weight is within the target product's molecular weight range, that it shows a blue color derived from the bleomycin-copper complex, that 6N hydrochloric acid 110
℃ for 18 hours, the above-mentioned specific amino acids derived from bleomycin, such as β-hydroxystidine, are detected at the same time as normal amino acids derived from proteins or polypeptides. I can confirm that I have it. More surprisingly, the present substance, for example, 3-((S)-1-phenylethylamino)propylaminobleomycin-BSA conjugate (copper-containing body) inhibited the proliferation of HeLaS ₃ cells. Its ID ₅₀ value was 57 μg/ml. This fact suggests that if a carrier is selected that has cancer specificity in its protein portion, it will be possible to selectively transport bleomycin to cancer tissues and attack cancer cells. In addition, active ester compounds of the general formula [] can bind to proteins under mild conditions of being neutral and below room temperature, so when an enzyme is used as the protein, there is less loss of enzyme activity, so this method The bleomycin-enzyme conjugate prepared by
It can be used as a labeled antigen in EIA. The enzyme to be bound may be any enzyme having an amino group that is not involved in the activity, and examples thereof include peroxidase, β-D-galactosidase, alkaline phosphatase, glucose oxidase, and lysozyme. Next, the present invention will be specifically explained using examples. Example 1) Synthesis of compound represented by general formula [] a) -((S)-1-phenylethylamino)
500 mg of propylaminobleomycin dihydrochloride (copper-containing substance) was dissolved in 50 ml of methanol, 66 mg of sodium glyoxylate hydrate was added, and then 20 mg of sodium cyanoborohydride was added. After reacting at 40℃ for 20 hours,
Lower the pH after the reaction to 1.0 with a 6N aqueous hydrochloric acid solution,
The reaction was increased by leaving it for 10 minutes. After neutralizing with 1N sodium hydroxide, methanol was distilled off under reduced pressure, and distilled water was added to the residue to make 10 ml. A column (100 ml) filled with CM Sephadex C-25 (Na+ type: Pharmacia, manufactured by Fine Chemical Co., Ltd.) equilibrated with 1/20 molar phosphate buffer at pH 6.8 in advance.
was injected into and adsorbed. Elute by the linear concentration gradient method in which the sodium concentration is gradually increased to 1.0 mol by continuously adding sodium chloride to the above buffer solution, collect 50 ml of the blue fraction that elutes at around 0.1 to 0.15 mol, and pre-prepare with distilled water. (100 ml volume) was injected into a column of Amberlite XAD-2 (manufactured by Rohm and Haas) packed with 100 ml of Amberlite, and the target substance was adsorbed.
After cleaning the column with 150ml of distilled water, 1/50M
Elution was performed with an aqueous hydrochloric acid solution-methanol (1; 4 v/v). The blue fractions were collected and neutralized with an anion exchange resin, Dowex 44 (OH type: manufactured by The Dow Chemical Company), and then concentrated under reduced pressure and freeze-dried to obtain the blue color of Compound No. 1. 270 mg of powder was obtained. The melting point of this product is 210-212℃ (decomposition), and the maximum ultraviolet absorption measured with distilled water is 292mμ, E1
%/1cm was 118.7. The infrared absorption large wavenumber (cm ^-1 ) measured by the potassium bromide tablet method is 3400,
1720, 1640, 1550, 1460, 1370, 1330,
They were 1130, 1100, 1060, 1010, 980, 920, 760. Other physical and chemical properties are shown in Table 2. b) - [N-Methyl-N-(3-aminopropyl)amino]propylaminobleomycin trihydrochloride (copper-containing compound) 1g was added to 100ml of 0.1N acetic acid potassium methanol solution - 0.1N acetic acid methanol solution (2:1v/ v) and added 57 mg of glyoxylic acid followed by 26 mg of sodium cyanoborohydride. After reacting at 40℃ for 24 hours, the pH of the reaction solution was adjusted with 6N hydrochloric acid aqueous solution.
was lowered to 1.0 and left for 10 minutes to stop the reaction.
After neutralizing with 1N sodium hydroxide, methanol was distilled off under reduced pressure, and distilled water was added to the residue to make 20 ml. This is done in advance at PH6.8,
It was injected into a column (100 ml volume) packed with CM Sephadex C-25 (Na+ type: Pharmacia, manufactured by Fine Chemical Co., Ltd.) equilibrated with 1/20 molar phosphate buffer and adsorbed. Elution was performed using a linear concentration gradient method in which the sodium concentration was gradually increased to 1.0M by continuously adding sodium chloride to the above buffer solution.
Collect 120ml of the blue fraction that elutes around the molar range,
Amberlite pre-filled with distilled water
The target product was adsorbed by injecting it into a column (100 ml) of XAD-2 (manufactured by Rohm and Haas). After cleaning the column with 150ml of distilled water,
1/50M hydrochloric acid aqueous solution - methanol (1:4v/
v). Collect the blue fraction and use an anion exchange resin, Dowex 44 (OH type;
After neutralizing with Dow Chemical Co.),
By concentrating and freeze-drying under reduced pressure, 630 mg of blue powder of Compound No. 5 was obtained. The melting point of this product is 207℃ (decomposition), and the maximum ultraviolet absorption measured with distilled water is 292mμ, E1%/1
cm was 100. The maximum infrared absorption wavenumber (cm ^-1 ) measured by the potassium bromide tablet method was 3400, 2950,
1730, 1640, 1580, 1560, 1460, 1400,
1380, 1330, 1140, 1100, 1070, 1020, 990,
It was 930. Other physical and chemical properties are shown in Table 2. In the above reaction, 3-[N-methyl-N-(3-
Using 1 g of glyoxylic acid, 140 mg of glyoxylic acid, and 52 mg of sodium cyanoborohydride, 510 mg of compound No. 6 was obtained.
The following compounds (Table 1) were obtained. The melting point of this product is 202-204℃ (decomposed), the maximum ultraviolet absorption measured in distilled water is 292mμ, E1%/
1 cm was 107.3. The maximum infrared absorption wavenumber (cm ^-1 ) measured by the potassium bromide tablet method is 3430,
2950, 1720, 1640, 1550, 1460, 1390,
1370, 1320, 1250, 1090, 1130, 1050,
It was 1010,990. Other physical and chemical properties are shown in Table 2.

[Table] 50 mg of compound No. 1 obtained in c) b)
was dissolved in 1 ml of dimethylformamide, and N
- adding 36 mg of hydroxysuccinimide;
Then dicyclohexylcarbodiimide 31mg
was added. After stirring and reacting at room temperature for 20 hours,
20 ml of acetone was added and the resulting precipitate was collected by filtration. The precipitate was washed with acetone, dried, dissolved in water, insoluble materials were filtered off, and the filtrate was freeze-dried to obtain 46 mg of blue powder of Compound No. 2 (Table 1). The maximum infrared absorption wavenumber (cm ^-1 ) of this product measured using the potassium bromide tablet method is
3400, 2950, 1820, 1780, 1730, 1640,
1550, 1460, 1370, 1240, 1210, 1100,
They were 1060, 1010, 920, 880, 810. Other physical and chemical properties are shown in Table 3. In the above method, instead of N-hydroxysuccinimide, p-nitrophenol or N
-Hydroxy5-norbornene-2,3-dicarboximide was used to synthesize Compound No. 3 and Compound No. 4, respectively. Their physical and chemical properties are shown in Table 3. d) 30 mg of compound No. 5 obtained in b)
was dissolved in a mixture of 1 ml of dimethylformamide and 0.05 ml of distilled water, and N-hydroxy-5
33 mg of -norbornene-2,3-dicarboximide was added, followed by 19 mg of dicyclohexylcarbodiimide. After stirring and reacting at room temperature for 15 hours, 20 ml of acetone was added and the resulting precipitate was collected by filtration. The precipitate was washed with acetone, dried, dissolved in water, filtered to remove insoluble matter, and the resulting solution was freeze-dried to obtain 30 mg of a blue powder of Compound No. 9. The maximum infrared absorption wavenumber (cm ^-1 ) of this product measured using the potassium bromide tablet method is
3430, 2950, 1780, 1740, 1720, 1650,
1580, 1560, 1460, 1380, 1320, 1240,
They were 1140, 1100, 1060, 1010. Other physical and chemical properties are shown in Table 3. Compound No. 7 was synthesized by the above method using N-hydroxysuccinimide instead of N-hydroxy 5-norbornene-2,3-dicarboximide. Their physical and chemical properties are shown in Table 3. Furthermore, in the above method, the compound
Using compound No. 6 instead of compound No. 5, N-hydroxysuccinimide,
N-hydroxy 5-norbornene-2,3-
Compound Nos. 8 and 10 were obtained by reacting with each dicarboximide. Their physical and chemical properties are shown in Table 3. Table 3 Compound number Thin layer chromatography Rf value *1 2 0.46 3 0.15 4 0.45 7 0.89 8 0.86 9 0.82 10 0.81 *1: Silica gel 60F254, Silanized (Merck & Co.), 20% ammonium acetate-methanol (50:50v/ (v%) However, for compound 6), the value in parentheses is shown in 20% ammonium acetate-methanol (70:30v/v%). 2) Synthesis of protein conjugate of bleomycin a) 2.5ml of 53mg of bovine serum albumin (BSA)
198 mg of Compound 2 obtained in 1) B) above (bleomycin/BSA molar ratio = 1:175) was added, dissolved in 0.05M phosphate buffer (PH7.5). After reacting overnight at room temperature, add 150 ml of Sephadex, which has been equilibrated with the above buffer.
Inject into a column packed with G-50, perform chromatography with the same buffer, and collect the eluate.
It was fractionated into 2.5 ml portions. Blue bands due to copper bleomycin were eluted in fractions 18-24 and 48-65, respectively. Fractions 18 to 24 simultaneously exhibited protein-derived absorption at 280 nm, indicating the formation of a conjugate. Fractions 18 to 24 were dialyzed against water overnight and then lyophilized to obtain 43 mg of a blue powder of bleomycin-BSA conjugate. E1%/1cm of this material at 280nm is
It was 23.9. BSA, and bleomycin
When the number of bonds was calculated using the respective E1% values at 280 nm, 6.6 and 105.6, 8.4 molecules of bleomycin were bound per 1 molecule of BSA. Also, 32mg of compound 2 for 53mg of BSA
(Bleomycin/BSA molar ratio = 1:25) was used to carry out the reaction, and 40 mg of the conjugate was obtained. E1%/1cm of this product at 280nm is 12.61
2.6 molecules of bleomycin were bound per molecule of BSA. b) In the same manner as in 2)-a), 23 mg of BSA was reacted with 23 mg of compound 9, and as a result of purification, 21 mg
The conjugate was obtained. At 280nm of this
E1%/1cm was 3.31, and according to the same calculation as in a), 15.1 molecules of bleomycin were bound per molecule of BSA.

Claims (1)

[Claims] 1 General formula [BX] -NH-A-NR _2-o -(CH ₂ Y) _o [In the formula, [BX] represents a residue obtained by removing the hydroxyl group from the carboxyl group of bleomycin acid, A is a lower alkylene or an alkylene bonded through a nitrogen atom, R is H or an alkyl substituted with alkyl or phenyl, Y is a carboxyl group or a reactive derivative thereof, and n is 1 or 2. A method for producing a bleomycin protein or polypeptide conjugate, which comprises condensing the represented bleomycin derivative with a protein or polypeptide.