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Modified arginine deiminase

A technology of arginine deiminase and arginine, applied in hydrolytic enzymes, medical preparations containing active ingredients, peptide/protein components, etc., can solve the problems of immunogenic toxicity, unstable linkers, etc.

Inactive Publication Date: 2004-10-13
上海复旦张江生物医药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The presence of this linking group is not necessary, and the presence of the linking group (1) may provide a target site for enzymatic or hydrolysis, resulting in instability of the linker, such as the ester bond in SS-PEG; (2) Linking groups that may be immunogenic or potentially toxic, such as cyanuric chloride

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Production of recombinant ADI

[0072] The gene of arginine deiminase is synthetic according to the sequence of Mycoplasma arginini published in Infect.Immun., 58:3788-3795 (1990) such as T.Ohno, and open reading frame comprises 1230 base pairs (Fig. 1 and SEQ ID NO: 1), wherein 5 codons TGA encoding tryptophan are unintentional codons of Escherichia coli and changed to TGG. SEQ ID NO: 1 encodes a 410 amino acid arginine deiminase (shown in SEQ ID NO: 2 and Figure 2).

[0073] The expression and renaturation in Escherichia coli were carried out according to the following method: Specifically, the synthetic ADI gene was inserted into the SpeI and BamHI sites of pET32 vector (Novagen Company), and the expression plasmid pET32-ADI was constructed. pET32-ADI was transformed into conventional Escherichia coli strain BL21, and the transformed bacteria were cultured in 500ml LB medium, and the expression was induced with 1.0mM IPTG for 4 hours. Bacteria were disrupted by ult...

Embodiment 2

[0076] ADI cross-linked with TMPEG

[0077] The ADI prepared in Example 1 and TMPEG with an average molecular weight of 5000 Da were used in this example.

[0078] The cross-linking reaction of PEG and ADI was carried out at pH 7.5, 50 mM phosphate buffer solution with 0.125 M sodium chloride added, the mass ratio of PEG and ADI was 30:1, and stirred at room temperature for 2 hours. After the reaction, unbound PEG in the mixture was removed by ultrafiltration membrane dialysis, and the degree of modification and enzyme activity of the conjugate were measured.

[0079] The following assay was used to determine the degree of modification of arginine deiminase:

[0080] Carry out with reference to the method described in J.Biol.Chem., 252.3582-(1977) by A. Abuchowski et al., that is, titrate the free amino group with the trinitrobenzenesulfonic acid (TNBS) method, and determine the modification by comparing the change of the free amino group before and after PEG modification. d...

Embodiment 3

[0086] Cross-linking of ADI with mPEG-C1

[0087] The ADI prepared in Example 1 and mPEG-Cl with an average molecular weight of 5000 Da were used in this example.

[0088] The cross-linking reaction between mPEG-Cl and ADI was carried out in 20 mM phosphate buffer at pH 7.0, the molar ratio of PEG to ADI was about 40:1, and stirred at room temperature for 2 hours. After the reaction, unbound PEG in the mixture was removed by ultrafiltration membrane dialysis, and the degree of modification and enzyme activity of the conjugate were measured. It was purified by Sephacryl S-300HR (Pharmacia) gel chromatography, and fractions with a molecular weight of about 200,000 Da were collected. In this example, after removing unbound PEG, the total modification degree of the conjugate was 53%, and the remaining enzyme activity after modification accounted for 55% of the initial enzyme activity.

[0089] The conjugate was further purified by Sephacryl S-300HR (Pharmacia) gel chromatography...

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Abstract

The present invention relates to a modified arginine deiminase having no connecting gene, it is a compound with formula (1) structure, said formula (1) is ADI-(PEG)n (I): in which PEG represents polyethylene glycol whose average molecular weight is 1000-20000 Da, ADI represents arginine deiminase, and 'represents covalent bond between PEG and ADI, and n is integral number of 2-30. Said invention also provides preparation method of modified arginine deiminase and its correspondent medicine composition. Said invented compound has obvious effect for resisting tumor, more stable chemical property and lower immunogenicity.

Description

technical field [0001] The invention relates to arginine deiminase modified with polyethylene glycol and its application in treating tumors. Background technique [0002] China is one of the countries with a high incidence of primary liver cancer, with an annual incidence of 5-10 per 100,000 population. About 130,000 people die from primary liver cancer every year, accounting for the third place among all malignant tumors. In other countries and regions in Asia, such as Japan and Taiwan, liver cancer is also one of the main forms of cancer. The development of drugs for the treatment of liver cancer is very urgently needed and of great significance. [0003] Tumor cells and normal cells have different nutritional requirements. Normal cells can synthesize certain non-essential amino acids independently, while tumor cells lose the ability to synthesize such amino acids. Using enzymes that can degrade this type of amino acid to selectively "starve" tumor cells is theoretically...

Claims

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Application Information

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IPC IPC(8): A61K38/46A61K38/50A61P35/00C12N9/14C12N9/78
Inventor 张文伯苏勇
Owner 上海复旦张江生物医药股份有限公司
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