A bioengineered arginine-depleting enzyme as a long-lasting therapeutic agent against cancer

• Published in: Applied microbiology and biotechnology 2020-05, Vol.104 (9), p.3921-3934
• Main Authors: Chung, Sai-Fung, Kim, Chi-Fai, Tam, Suet-Ying, Choi, Man-Chung, So, Pui-Kin, Wong, Kwok-Yin, Leung, Yun-Chung, Lo, Wai-Hung
• Format: Article
• Language: English
• Subjects: Animals; Arginase; Arginase - genetics; Arginase - therapeutic use; Arginine; Arginine - antagonists & inhibitors; Arginine deiminase; Bioengineering; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Cancer; Cancer therapies; Carbon dioxide; Care and treatment; Cell Line, Tumor; Chemical compounds; Circular dichroism; Clinical trials; Cobalt; Depletion; Dichroism; Enzymatic activity; Enzyme activity; Enzyme kinetics; Health aspects; Heavy metals; Humans; Immunogenicity; Injection; Ions; Life Sciences; Manganese; Metal ions; Metals; Methods; Mice; Mice, Inbred BALB C; Microbial enzymes; Microbial Genetics and Genomics; Microbiology; Mutants; Mutation; Neoplasms - drug therapy; Nickel; Pharmacodynamics; Pharmacology; Physiological aspects; Protein Engineering; Protein Structure, Secondary; Reaction kinetics; Spectrometry; Tumor cell lines
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-020-10484-4

l -Arginine (L-Arg) depletion has attracted great attention in cancer therapy. Although two types of arginine-depleting enzymes, arginine deiminase (ADI) and human arginase I, are undergoing clinical trials, random site of PEGylation, low efficacy of heavy metal as co-factor, and immunogenicity limit the performance of these drugs and cause difficulty in a homogeneous production. Here we screened ten catalytic metal ions and have successfully produced a site-specific mono-PEGylated human arginase I mutant by conjugating the Cys 45 residue to PEG-maleimide to minimize the decrease in activity and produce a homogeneous product. The catalytic efficiency trend of metal ion–enriched human arginase I mutant (HAI) was Co 2+  > Ni 2+  ≫ Mn 2+ . The overall k cat /K M values of Co-HAI and Ni-HAI were higher than Mn-HAI by ~ 8.7- and ~ 5.2-folds, respectively. Moreover, the results of enzyme kinetics and circular dichroism spectrometry demonstrated that the 20 or 40 kDa linear and branched PEG attached on the HAI surface did not affect the enzyme activity and the protein secondary structures. In vitro studies showed that both Co-HAI-PEG20L and Ni-HAI-PEG20L inhibited the growth of eight types of cancer cell lines. The pharmacodynamic study in mice demonstrated that the i.p. administration of Co-HAI-PEG20L at 13 mg/kg and Ni-HAI-PEG20L at 15 mg/kg was able to maintain a L-Arg level below its detection limit for over 120 h after one injection. The body weights of mice could return to normal levels within 5 days after injection, showing that the doses were well-tolerated. Therefore, both the Ni-HAI-PEG20L and Co-HAI-PEG20L are promising candidates for cancer therapy. Key Points • Mono-PEGylation applied on human arginase I mutant (HAI) successfully. • The catalytic efficiency of Co- and Ni-enriched HAI was higher than the wild type. • At least eight types of cancer cell lines were inhibited by Co- and Ni-HAI-PEG20L. • Co- and Ni-HAI-PEG20L were able to achieve weekly depletion of L-Arg. Graphical abstract