TY - JOUR
T1 - Nonclinical Evaluation of Single-Mutant E. coli Asparaginases Obtained by Double-Mutant Deconvolution
T2 - Improving Toxicological, Immune and Inflammatory Responses
AU - Ruiz-Lara, Grace
AU - Costa-Silva, Tales A.
AU - Muso-Cachumba, Jorge Javier
AU - Cevallos Espinel, Johanna
AU - Fontes, Marina Gabriel
AU - Garcia-Maya, Mitla
AU - Rahman, Khondaker Miraz
AU - Rangel-Yagui, Carlota de Oliveira
AU - Monteiro, Gisele
N1 - Publisher Copyright:
© 2024 by the authors.
PY - 2024/6
Y1 - 2024/6
N2 - Acute lymphoblastic leukaemia is currently treated with bacterial L-asparaginase; however, its side effects raise the need for the development of improved and efficient novel enzymes. Previously, we obtained low anti-asparaginase antibody production and high serum enzyme half-life in mice treated with the P40S/S206C mutant; however, its specific activity was significantly reduced. Thus, our aim was to test single mutants, S206C and P40S, through in vitro and in vivo assays. Our results showed that the drop in specific activity was caused by P40S substitution. In addition, our single mutants were highly stable in biological environment simulation, unlike the double-mutant P40S/S206C. The in vitro cell viability assay demonstrated that mutant enzymes have a higher cytotoxic effect than WT on T-cell-derived ALL and on some solid cancer cell lines. The in vivo assays were performed in mice to identify toxicological effects, to evoke immunological responses and to study the enzymes’ pharmacokinetics. From these tests, none of the enzymes was toxic; however, S206C elicited lower physiological changes and immune/allergenic responses. In relation to the pharmacokinetic profile, S206C exhibited twofold higher activity than WT and P40S two hours after injection. In conclusion, we present bioengineered E. coli asparaginases with high specific enzyme activity and fewer side effects.
AB - Acute lymphoblastic leukaemia is currently treated with bacterial L-asparaginase; however, its side effects raise the need for the development of improved and efficient novel enzymes. Previously, we obtained low anti-asparaginase antibody production and high serum enzyme half-life in mice treated with the P40S/S206C mutant; however, its specific activity was significantly reduced. Thus, our aim was to test single mutants, S206C and P40S, through in vitro and in vivo assays. Our results showed that the drop in specific activity was caused by P40S substitution. In addition, our single mutants were highly stable in biological environment simulation, unlike the double-mutant P40S/S206C. The in vitro cell viability assay demonstrated that mutant enzymes have a higher cytotoxic effect than WT on T-cell-derived ALL and on some solid cancer cell lines. The in vivo assays were performed in mice to identify toxicological effects, to evoke immunological responses and to study the enzymes’ pharmacokinetics. From these tests, none of the enzymes was toxic; however, S206C elicited lower physiological changes and immune/allergenic responses. In relation to the pharmacokinetic profile, S206C exhibited twofold higher activity than WT and P40S two hours after injection. In conclusion, we present bioengineered E. coli asparaginases with high specific enzyme activity and fewer side effects.
KW - bioengineer
KW - E. coli asparaginase
KW - leukaemia
KW - nonclinical assays
UR - http://www.scopus.com/inward/record.url?scp=85195883325&partnerID=8YFLogxK
U2 - 10.3390/ijms25116008
DO - 10.3390/ijms25116008
M3 - Article
AN - SCOPUS:85195883325
SN - 1661-6596
VL - 25
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 11
M1 - 6008
ER -