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

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...

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

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