JPH04164098A - Chemically modified, granular spherical colony stimulus factor derivative - Google Patents

Abstract

PURPOSE: To obtain the chemically modified subject protein with extended blood retention time and long-lasting pharmacological activity, by linking polyeth ylene glycol to a human G-CSF polypeptide derivative wherein Cys at the 17-po sition is substituted by other amino acids.

CONSTITUTION: A polypeptide having the amino acid sequence of the formula (Xaa is an amino acid residue except Cys; Met may further be bonded to the tip of the N-terminal) which has been produced by a transformant obtained by transforming a host such as E. coli, animal cell, etc., in gene recombinant technique and has been separated and purifed, is reacted with an activated polyethylene glycol, e.g. methoxypolyethylene glycol succinidimylsuccinate, thereby to obtain the objective chemically modified protein. Example of the amino acid except Cys is preferably Ala and it is preferred to use polyethylene glycol with a molecular weight of 4,000-20,000. The resultant modified product is improved in its thertmostability and the yield in the modification is good.

COPYRIGHT: (C)1992,JPO

Description

[Detailed description of the invention]

[0001]

[Industrial application field]

This invention relates to the chemical modification of granulocyte colony stimulating factor (G-C3F) polypeptide derivatives with amino acid substitutions, which modifications can alter the chemical and/or physiological properties of G-C3F. . [0002]

[Conventional technology]

h) G-C3F is one of the hematopoiesis-promoting factors and has been shown to exist in the culture medium of human bladder cancer cell line 5637 (ATCCHT8-9) (Welt et al.: P
roc, Natl, Acad, Sci, U, S
, A, 82.1526-1530. (1985))
. Furthermore, the DNA sequence encoding this gene has been determined (Japanese Patent Publication No. 63-500636), making it possible to produce G-C3F by genetic recombination. [0003] G-C3F is effective for the treatment of normal hematopoietic disorders, the treatment of hematopoietic disorders caused by chemotherapy or radiation therapy, the treatment of fever during bone marrow transplantation or wound healing, and the treatment of bacterial inflammation (Werth et al. ; previously mentioned). Intercostal +4-164098 (3) [0004] On the other hand, when a physiologically active protein is administered, its medicinal efficacy may only be obtained for a short period of time due to its rapid clearance in the body. Furthermore, if the protein is highly hydrophobic, problems may arise in its stability. [0005] A method of modifying physiologically active proteins with polyethylene glycol is known for the purpose of extending the blood residence time, improving stability, or eliminating antigenicity. For example, JP-A No. 62-289522 discloses a reduction in the immunogenicity of TNF modified with polyethylene glycol or the like. In addition, in Japanese Patent Publication No. 62-503171, IL-2 modified with polyethylene glycol, etc.
Reduced aggregation of FN-β in aqueous solutions, increased blood half-life, and reduced immunogenicity are disclosed. In addition, plasminogen activator (JP 63-60938), IL-2, IFN-γ, SOD (JP 63-108
00) and IAP (Japanese Patent Application Laid-open No. 63-126900),
It has been disclosed that polyethylene glycol modification can extend blood half-life or eliminate antigenicity and immunogenicity. Also, G
-C3F has also been reported to have improved storage stability, extended blood retention time, etc. by modification with polyethylene glycol (Japanese Unexamined Patent Publication No. 1-316400). [0006]

[Problem to be solved by the invention]

H) When G-C3F is administered into the body, it is desired to extend the retention time in the blood in the body, thereby further increasing the durability of the expected drug effect, and improving thermal stability and yield. It is rare. [0007]

[Means to solve the problem]

In order to solve these problems with human G-C3F, the present inventors have made repeated studies and have developed a human G-C5F polypeptide derivative (hereinafter simply referred to as "G-C3F") in which cysteine at position 17 is replaced with another amino acid. The inventors have discovered that the above problems can be solved by bonding polyethylene glycol to a C3F derivative (referred to as "C3F derivative"), and have arrived at the present invention. [0008] [Specific Description] The G-C3F derivative of the present invention is produced from a transformant obtained by transforming a host such as Escherichia coli or animal cells by genetic recombination, and is obtained by isolation and purification. Any one can be used as long as it is available. However, among them, the amino acid sequence of SEQ ID NO: 1 is available in homogeneous quantities with good purity (however, Met may be further bonded to the beginning of the N-terminal side, and the 17th Xaa is Cy
Particularly preferred are G-C3F derivatives produced by recombinant E. coli having an amino acid other than s. Furthermore, a derivative (G-C
SF-A1a17) is preferred. [00093] In the present invention, the human G-C3F derivative and polyethylene glycol are preferably covalently bonded to each other via amino acid residues of the polypeptide. The residues are any reactive amino acids having free amino groups, etc., and the reactive groups of activated polyethylene glycol are linked to these free amino groups. Amino acid residues having free amine groups include lysine or N-terminal amino acid residues. [0010] The molecular weight of the polyethylene glycol used is not limited to a specific one, but typically 500 to 30,000, preferably about 4,000 to 20,000. [0011] Polyethylene glycol is attached onto the human G-C5F derivative via a terminal reactive group (spacer). Polyethylene glycol having a spacer is referred to as activated polyethylene glycol. Examples of spacers include those that mediate the bond between free amino groups and polyethylene glycol. As an activated polyethylene glycol that binds to a free amino group, for example, it is represented by the following formula, [0012]

embedded image [0013] Methoxypolyethylene glycol succinimidyl succinate, which is obtained by activating a succinate ester of methoxypolyethylene glycol with N-hydroxysuccinimide, is used. [0014] Alternatively, activated polyethylene glycol (2,4-bis(O-methoxypolyethylene glycol)-6-chloro-3-) riazine shown in the following formula synthesized from polyethylene glycol monomethyl ether and cyanuric chloride is used. [0015]

[0016] The covalent modification reaction may be carried out by any suitable method commonly used to react biologically active materials with activated polyethylene glycols. human GC
When the reactive amino acid on the 3F derivative is an amino acid residue having a free amino group, the pH is preferably 7.5-5.
10.0. The reaction is carried out in a buffer such as phosphate or borate at pH 7.5 to 10.0 and at a temperature of 4.
Carry out 1-5 hours at ~37°C. 1 to 2 active polyethylene glycols are added to the free amino group of human G-C3F.
00 times the molar amount, preferably 5 to 50 times the molar amount. [0017] The modification rate of amino acid residues can be freely varied depending on the amount of the above-mentioned activated polyethylene glycol used. [0018] If desired, the reaction solution is purified by a conventional protein purification method such as dialysis, salting out, ultrafiltration, ion exchange chromatography gel filtration, or electrophoresis to obtain the desired polyethylene glycol-modified human G-C5F. derivatives can be obtained. Ion exchange chromatography is particularly effective in removing polyethylene glycol and unmodified human G-C3F derivatives. Furthermore, if desired, a polyethylene glycol-modified human G-C3F derivative having a uniform degree of modification (number of bonds) can be purified by using ion exchange chromatography, gel filtration chromatography, or the like. [0019]

[Effect]

The polyethylene glycol-modified H)G-C3F derivative of the present invention has been recognized to have remarkable effects such as increased thermal stability and improved yield over polyethylene glycol-modified G-C5F. Polyethylene glycol-modified human G of the present invention
-C3F derivatives have essentially the same biological activity as unmodified H)G-C3F derivatives as well as unmodified H)G-C3F, and therefore have similar uses as unmodified H)G-C3F. It is effective for That is, due to its biological activity of increasing neutrophils, it is effective in the treatment of normal hematopoietic disorders, in the treatment of hematopoietic disorders caused by chemotherapy or radiotherapy, in bone marrow transplantation, or in the treatment of infectious diseases. [0020] The polyethylene glycol-modified H)G-C3F derivative is
By blending with medically acceptable diluents, tonicity agents, pH adjusters, etc., it can be used as a preparation that can be administered to patients. [0021] The administration method of a preparation containing a polyethylene glycol-modified human G-C3F derivative as an active ingredient may vary depending on the therapeutic purpose, but may be administered by subcutaneous, intramuscular or intravenous injection, or orally. The dosage can be determined according to the target disease and the patient's condition, but in the case of injection, the human G-C5F derivative weight is usually 0.1 μg to 5 mg per adult, and the same for oral administration. <0.
1 mg to 5 g can be administered. [0022]

【Example】

The present invention will be specifically explained in the following Examples, but the present invention is not limited thereto. [0023] A human G-C5F derivative in which the 17th cysteine was replaced with alanine (hereinafter referred to as [human G-C5F
-CSF-Ala17 J) was targeted for modification. For the modification, polyethylene glycol was used as a raw material, and methoxypolyethylene glycol succinimidyl succinate (manufactured by NOF Corporation) (activated PEG1) with an average molecular weight of about 4.500 was obtained by activating its succinate ester with N-hydroxysuccinimide. ) was used. [0024] Human G-CS F-A1a17 was combined with activated PEGI in 0.25 M sodium borate buffer (pH 8.0).
The reaction was carried out at ℃ for 1 hour. The amount of activated PEG1 is
A 40-fold amount was used relative to the free amino groups in C3F-A1a17. The products were purified using DEAE first exchange chromatography after buffer exchange with 5ephadex G 25 pre-equilibrated with 10mM NHHCO to form various forms of polyethylene glycol modified G-C3.
F-Ala17 (PEG modified G-CS F-Ala
17) as reagent and unreacted G-CS F-A as necessary.
It was isolated from 1a17. The PEG-modified G-C3F derivative obtained in this reaction was converted into PEG (4500) G-
It is called CSF-Ala17. [0025] The reactants were prepared according to the method of Laemml i (Nature,
227 pp. 680 (1970)), 5DS-PAGE was performed and CBB staining was performed. After staining, each lane of each gel was scanned. Takashi chromatography scanner (C3-930) was used for measurement.
was used. The average molecular weight determined from the scanning results was 47, and its distribution was 26K (18%), 34K
(31%), 54K (31%) and 74K (2
0%). In addition, these are) G-CS F
-It is a mixture of one to multiple molecules of activated PEG bound to Ala17, and can be further separated depending on the degree of modification (number of bonds) by using ion exchange chromatography or gel filtration chromatography. can. [0026] The human G-CS F-A1a17 to be modified was prepared according to Example 1.
This is the same as shown in . For the modification, activated polyethylene glycol (manufactured by Seikagaku Corporation) (referred to as activated PEG2) with an average molecular weight of 10.000 shown in Chemical formula 2 was prepared by converting the raw material polyethylene glycol into polyethylene glycol monomethyl ether and reacting it with cyanuric chloride. used. [0027] 1720 mg of human G-CS F-Ala was added to 0.0 mg of human G-CS F-Ala. Activated P in IM sodium borate buffer pH 10,0)
It was reacted with EG2 at room temperature for 1 hour. Regarding the amount of activated PEG, 5 times the amount of free amino groups in G-C3F was used. The product was buffer exchanged with 5ephadex G 25 equilibrated with 10mM NH4HCO3, and then PEG-modified human G-CS F-A1a17 was separated from the reagent and unreacted human G-CS using DEAE ion exchange chromatography. separated from F-Ala17,
PEG-modified human G-C5F-Ala17 2.3 mg
(yield: 11.5%). At the same time, human G-C5F was reacted with activated PEG2, separated, and PEG-modified G-C5F
1. Omg (yield 5.0%) was obtained. From this result, human G-CS F-Ala17
It was found that the PEG-modified product could be obtained in a higher yield than 3F. [0028] The PEG-modified human G-C5F derivative obtained in this reaction was hereinafter referred to as PEG (10000)G-CS F-Ala1.
It's called 7. As in Example 1, 5DS-
When the molecular weight was estimated on PAGE, it was found that P
The average molecular weight of G (10000) G-CS F -A1a17 is 51, and its distribution is 3QK (23
%), 42K (25%), 59K (27%) and 72K (25%). In addition, these are human G
-CS F-Ala17 with one to multiple molecules of active PE
It is a mixture of G-bonded compounds, and was further separated according to the degree of modification (number of bonds) using ion exchange chromatography or gel filtration chromatography. [0029] 175 mg of human G-CS F-Ala was reacted with activated PEG2 under the same conditions as in Example 2, and 5 ephad
PEG (10000) G-CS using ex G 25 and DEAE ion exchange chromatography.
F-Ala17 was obtained. TSK gel-G3000SWXL (
(manufactured by Tosoh Corporation) (7.5mmXmmX6O, flow rate 1m)
The l/min was huge. [0030] PEG2 trimolecular conjugate i form) was eluted around a retention time of about 15 minutes (yield 0, 13 mg, yield 2.6%).
), PEG2 bimolecular conjugate) has a retention time of approximately 17
(yield 0.16 mg, yield 3.2
%), then the PEG2-molecule conjugate (Mon. body) was eluted around a retention time of about 18 minutes (yield 0
．． 12 mg, yield 2.4%). The obtained chemically modified product is M
5DS-polyacrylamide gel shows that in the ono form, one molecule of PEG2 is bound to the human G-CS F-Ala17-molecule, in the Di form it is bound to a binding molecule, and in the Tri form it is bound to a binding molecule. Confirmed by electrophoresis. The purity of each was 90% or more. JP-A-4-164098 (1Q) [0031] PEG (4500) G prepared in Example 1.2
-CSF-A1a17 and PEG (10000
) The pharmacological effects of G-CS F-Ala17 in mice were investigated. Samples were administered intravenously at a dose of 10 μg protein/kg to mice (ICR (male)) at the age of 7 weeks, and blood was collected from the orbital venous plexus of the mice at 24.48 and 72 hours later. The white blood cell count was measured using Toa Medical Electronics. At the same time, the blood smear specimen was stained with light, and an automatic blood cell classification device (MICR) was used.
White blood cell fraction was measured using OX, Cubic Denki), and the number of neutrophils was determined. The results are shown in Table 1. [0032] P E G (4500) G-CS F -Ala1
7, and P E G (10000) G-CS F
-Ala17 increased the number of neutrophils compared to unmodified G-C3F and polyethylene glycol-modified G-C3F, and it was observed that the neutrophil increasing effect persisted up to 48 and 72 hours later. . In addition, PEG used for modification
It was estimated that the larger the molecular weight of the drug, the greater the pharmacological effect. [0033]

[Table 1] i of PEG-modified G-CS F-A1a17
n vivo pharmacological effect vehicle-C3F G-C3F-Ala17 PEG (4500) G-C3F-Ala17PEG
(10000) G-C3F-A1a179.4 (1,5
11,7(1,2 22,3(3,411,3(2,1 24,2(2,810,8(1,7 25,6(6,118,0(2,8 87,0( 7,144,9(4,2 11,7(1,8 9,7(0,9 8,0(0,7 15,1(1,0 17,0(1,2) *Dose: 10μg protein /Kg*Dispersion of mice: Average of 6 mice each (standard deviation) PEG prepared in Example 2 (10000) G-
The thermal stability of CSF-Ala17 was determined by PEG (100
00) compared with G-CS F. each sample at a concentration of 0.1
10 mM phosphate buffer (p
After dissolving in H7,0) and heating at 53°C for 6 hours, the residual activity of G-C5F in the solution was measured. The activity of G-C3F was determined by the method of P. Ralph et al. (Brad (1986) 68.
633-639) and the method of R.N. Moore et al. (Journal of Immunology (1983) 131.23
74-2378) to mouse bone marrow cells.
- Thymidine incorporation was measured as an index. The results are shown in Table 2. Residual activity (%) is a relative proportion to the initial activity before heating, and is defined by the following formula. [0034] [Equation 1] Residual activity (%) = (Activity after heating) / (Initial activity) xlOOP E G (10000) G-CS
F-A1a17ba, P E G (10000)
It was confirmed that the residual activity was higher and the thermal stability was improved compared to G-CSF. [0035]

[Table 2] PEG (10000)G-C3F-Ala
Thermal stability of 17 (53℃, 6hr) PEG (1000
0) G-C3F 8P E G
10000) G-CS F-A1a17
61 (10 mM phosphate buffer, pH 7.0) Reference A G-C3F derivative (G-CS F-Ala17) in which cysteine at position 17 was replaced with alanine was obtained as follows. [0036] Plasmid pSA2116 containing DNA encoding the above G-C3F derivative (Japanese Patent Application Laid-Open No. 2-104598
) carrying Escherichia coli (p S A2116/ AM 7
) was cultured with shaking overnight at 28°C in a medium containing ampicillin and kanamycin. 5ml of this culture solution
00ml of L medium and cultured with shaking at 28°C for 4 hours. 500 ml of a medium that had been preheated to 60°C was added to the culture solution, and the temperature was raised to 42°C and cultured with shaking for an additional 3 hours. [0037] The culture solution was further centrifuged to obtain about 20 g of bacterial cells. 14
After adding and suspending 0ml of distilled water, IMDTT was added to a final concentration of 1mM. During this time, the solution was kept at 4°C. The bacterial cells were crushed using a French press, and the precipitate was collected. From this precipitate, Tanaka et al.'s method [H, Tanaka et al.
et al.: The Journal of Pharmac.
ology and ExperimentalThe
rapeutics 251.1199 (1990)
] The G-C5F derivative was extracted, purified, solubilized, and regenerated. [0038] Effects of the Invention From the above results, the polyethylene glycol-modified H)G-C5F derivative provided by the present invention has a lower blood concentration by half compared to unmodified G-C3F and unmodified G-CS F-Ala17. The drug's efficacy (neutrophil increase effect) continues for a longer period of time, making it possible to administer smaller doses and fewer times when administered in vivo. Furthermore, the polyethylene glycol-modified human G-C5F derivative of the present invention had an improved yield and was also found to have improved thermal stability compared to polyethylene glycol-modified G-C5F. It is expected that the effects of these inventions can further contribute to treatment using H) G-C3F. [0039]

[Sequence list]

Sequence number: 1 Sequence length = 174 Sequence type Niamino acid Topology: Linear Sequence type: Peptide sequence Thr Pro Leu Gly Pro Ala S
er Ser Leu Pro Gln Ser Ph
e Leu Leu Lysl 5
10 15Xaa L
eu Glu Gin Val Arg Lys Il
e Gln Gly Asp Gly Ala Ala
Leu GlnGlu Lys Leu Cys A
la Thr Tyr Lys Leu Cys Hi
sLeu Leu Gly His Ser Leu
Gly Ile Pro Trp AlaPro Se
r Gin Ala Leu Gln Leu Ala
Gly Cys LeuGly Leu Phe L
eu Tyr Gln Gly Leu Leu Gl
n AlaPro Glu Leu Gly Pro
Thr Leu Asp Thr Leu G1nPh
e Ala Thr Thr Thr Ile Trp Gln
Gin Met Glu GluAla Leu G
ln Pro Thr Gln Gly Ala Me
t Pro AlaGin Arg Arg Ala
Gly Gly Val Leu Val Ala 5
erLeu Glu Val Ser Tyr Arg
Val Leu Arg His LeuPro G
lu Glu Leu Va1Pro Leu Ser
Ser CysSer Gln Leu His S
erLeu Glu Gly Ile SerLeu
Asp Val Ala AspLeu Gly Me
t Ala Pr. Phe Ala Ser Ala Phel40 His Leu Gln Ser Phel60 Ala Gln Pr.

Claims (3)

[Claims] Claim 1: Having the amino acid sequence of SEQ ID NO: 1 (however, Met may be further bound to the beginning of the N-terminal side, and the 17th Xaa refers to an amino acid other than Cys), and has an exogenous DNA sequence. A chemically modified protein formed by binding polyethylene glycol to a polypeptide characterized by being an expression product of a host cell. 2. The chemically modified protein according to claim 1, wherein the amino acid other than Cys is Ala. 3. The chemically modified protein according to claim 1 or 2, wherein the polyethylene glycol is bonded via an amino group of an amino acid of the polypeptide.