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Methoxypolyethylene glycol-modified arginine deiminase, preparation thereof and use thereof

A technology of arginine deiminase and methoxy polyethylene glycol, which is applied in the field of arginine deiminase and can solve the problems of increasing the enzyme, uncertain anti-tumor efficacy, and lack of it.

Active Publication Date: 2009-12-02
JIANGSU T MAB BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even though some primary amine groups cannot react with active PEG due to steric hindrance, the number of total product species ultimately generated by the modification reaction is still surprising
If the special modification method in U.S. Patent No. 5,985,265 is used to bind PEG to the N-terminus of arginine deiminase, although relatively high activity can be maintained and a uniform product can be obtained, the N-terminal binding of PEG cannot completely shield arginine Immunogenic site of acid deiminase, still not available in clinical studies
Therefore, there are many disadvantages in the existing PEG-modified arginine deiminase: 1. Some primary amino groups are in the active site of arginine deiminase, which will seriously affect the biological activity after being modified, so Clinical use will inevitably increase the amount of enzyme used, but the increase in enzyme amount will increase immunogenicity; 2. Due to the uncertainty of the modification site, can the site with strong immunogenicity of arginine deiminase be shielded It is also uncertain; 3. Since the modified product is composed of a large number of mixtures, the structure and quantity of each modified product in the modified mixture cannot be determined by existing analytical methods, so there is no good quality control method to ensure the modification Quality Stability of the Arginine Deiminase Mixture
The final result brought about by these defects is that the PEGylated arginine deiminase obtained by the existing modification method is different between different batches, and people cannot predict its biological activity and immunogenicity, and it also lacks effective means to control
These deficiencies have been shown from the existing clinical trials of PEGylated arginine deiminase, such as uncertain antitumor efficacy, immunogenicity caused by repeated administration, and some serious toxic side effects caused by it , such as severe liver damage, allergic reactions, elevated lipase (Li-Jiuan Shen, Current Opinion in Molecular Therapeutics 8: 240-248(2006))
[0005] Many studies have proved that increasing the modification degree of protein or enzyme can increase the circulating half-life of the modification, but increasing the modification degree will reduce the biological activity of protein or enzyme (Rameshraja P et al., Current aspect inpharmacology of modified homoglobins[J] Adv drug deliv Review. 40, 185 (2000))
Generally, a low PEG modification rate has little effect on enzyme activity, but the half-life is short and there are few shielded epitopes, so it has little effect on reducing immunogenicity; a high PEG modification rate has a longer half-life, and the modified protein is blocked There are also many shielded antigenic determinants, so it has a great effect on reducing immunogenicity, but it also has a high impact on biological activity
To increase the modification rate, reduce immunogenicity, and prolong the half-life without reducing the biological activity of the modified enzyme is always an extremely difficult task under the current technical conditions

Method used

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  • Methoxypolyethylene glycol-modified arginine deiminase, preparation thereof and use thereof
  • Methoxypolyethylene glycol-modified arginine deiminase, preparation thereof and use thereof
  • Methoxypolyethylene glycol-modified arginine deiminase, preparation thereof and use thereof

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Experimental program
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Embodiment 1

[0055] Embodiment 1: the production of recombinant ADI

[0056] Many studies have shown that ADI from various mycoplasma sources has the same enzymatic properties although there are certain differences in sequence. Such as ADI derived from Mycoplasma arginini (ATCC23243) (SEQ ID NO: 1), Mycoplasma hominis (ATCC23114) (SEQ ID NO: 2), Mycoplasma arthritis (ATCC23192) (SEQ ID NO: 3), amino acid sequence homology More than 96%. Literature research (Clark, MikeA, U.S. Patent No.6183738) has proved that from specific activity, K m , V max , and pH optima prove that these enzymes are biochemically indistinguishable. ADI from these different sources are therefore suitable for use in the present invention. In the following examples of the present invention, ADI derived from Mycoplasma arginini (abbreviated as aADI) is used as an example for illustration.

[0057] The aADI gene is derived from arginine mycoplasma. Since some codons of mycoplasma cannot be effectively recognized and...

Embodiment 2

[0061] Embodiment 2.ADI enzyme activity assay

[0062] ADI uses arginine (Arg) as a substrate, and the product guanidine can react with blood and urine nitrogen reagent (BUN) at a high temperature of 100°C to appear red. Add the final concentration of 10mM Arg, 5μg ADI to the reaction system, add 20mM PB (pH 7.2) to 500ul, and react accurately at 37°C for 30min, take 50ul of the reaction solution and add it to the blood and urine nitrogen reagent (BUN), mix well, bathe in boiling water for 10min, and finally Measure OD 540 , with guanidine as the standard for the standard curve. The blank control was blood urine nitrogen reagent (BUN) without ADI. The content (μmol) of the reaction substrate guanidine was converted from the standard curve. Definition of enzyme activity unit: 1 enzyme activity unit (U) is defined as the enzyme activity that catalyzes the complete conversion of 1 μmol of arginine into 1 μmol of guanidine within 1 minute at 37°C.

[0063] The protein content ...

Embodiment 3

[0064] Example 3: PEG modification of raADI

[0065] Modification experiments were performed in two groups:

[0066] Reaction (1): in 100 milliliters of 1 mg / ml raADI solution (buffer system is 100mM Bicine, pH8.0), add PEG reagent (mPEG 20000 -SPA, Nektar). The reaction was stirred at room temperature for 1 hour. The modified product PEG-raADI was purified with Phenyl-Sepharose HP, and the corresponding target peak was collected, and finally removed with a G25 desalting column (NH 4 ) 2 SO 4 . The final prepared product is named mPEG 20000 -raADI.

[0067] Reaction (2): In addition to adding 10 mg / ml of BOC-Arg to the raADI solution first (corresponding to a molar ratio of BOC-Arg:raADI of 5:1), the purified modified product can be appropriately increased in the equilibrium stage before elution. Time, using the reversible binding and dissociation characteristics between the placebo substrate and the enzyme, it can be easily removed, other conditions are completely con...

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Abstract

The invention discloses a methoxypolyethylene glycol-modified arginine deiminase, preparation thereof and use thereof. As a gratuitous substrate is added in a modification reaction to protect active loci, the modified PEG-ADI has higher biological activity compared with the prior EG-ADI modified at the same modification rate. In addition, as specific affinity chromatography is used to remove immunogenic PEG-ADI, the finally prepared PEG-ADI has high activity, high immunologic uniformity and no in-vivo immunogenicity, thereby being more safe and effective in clinic.

Description

technical field [0001] The invention relates to arginine deiminase modified with methoxypolyethylene glycol, and its preparation and application. Background technique [0002] As early as 30 years ago, Storr and Burton proposed the possibility of controlling amino acids in tumor therapy (Storr JM, Burton AF. The effects of arginine deficiency on lymphoma cells. Br J Cancer. 30: 50-59 (1974)), Of these, the most studied is asparaginase, which breaks down asparagine needed for cell growth and maintenance of cell life. In many leukemia patients, leukemia cells are different from normal cells in that they cannot synthesize asparagine by themselves and must rely on asparagine provided by the outside world to maintain their survival. Treatment with asparaginase depletes the free asparagine needed by tumor cells in the body, resulting in tumor cell death, while normal cells are not much affected. However, L-asparaginase is derived from microorganisms and has strong immunogenicity...

Claims

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Application Information

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IPC IPC(8): C12N11/08A61K38/50A61K47/48A61P35/00A61K47/60
Inventor 凌建群裘霁宛温晓芳范敏王玉娇王叶飞梁伟峰
Owner JIANGSU T MAB BIOPHARMA
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