Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas

Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, ADI's application as anticancer drug is hampered by its low activity for arginine under physiological conditions mainly due to its high "K-M" (S-0.5) values which are often 1 magnitude higher than the arginine concentration in blood (0.10-0.12 mM arginine in human plasma). Previous evolution campaigns were directed by us with the aim of boosting activity of PpADI (ADI from Pseudomonas plecoglossicida, k(cat)=0.18 s(-1); S-0.5=1.30 mM), and yielded variant M6 with slightly reduced S-0.5 values and enhanced k(cat) (S-0.5=0.81 mM; k(cat)=11.64 s(-1)). In order to further reduce the S0.5 value and to increase the activity of PpADI at physiological arginine concentration, a more sensitive screening system based on ammonia detection in 96-well microtiter plate to reliably detect >= 0.005 mM ammonia was developed. After screening similar to 5,500 clones with the ammonia detection system (ADS) in two rounds of random mutagenesis and site-directed mutagenesis, variant M19 with increased k(cat) value (21.1 s(-1); 105.5-fold higher compared to WT) and reduced S-0.5 value (0.35 mM compared to 0.81 mM (M6) and 1.30 mM (WT)) was identified. Improved performance of M19 was validated by determining IC50 values for two melanoma cell lines. The IC50 value for SK-MEL-28 dropped from 8.67 (WT) to 0.10 (M6) to 0.04 mu g/mL (M19); the IC50 values for G361 dropped from 4.85 (WT) to 0.12 (M6) to 0.05 mu g/mL (M19).