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Chimeric and pegylated L-asparaginase: study of the conditions of expression, production and purification of an innovative anti-leukemic biopharmaceutical with low immunogenic potential

Grant number: 20/06982-3
Support type:Regular Research Grants
Duration: May 01, 2021 - April 30, 2023
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Cooperation agreement: Universidad de la Frontera
Principal researcher:Adalberto Pessoa Junior
Grantee:Adalberto Pessoa Junior
Principal researcher abroad: Jorge Gonzalo Farias Avendano
Institution abroad: Universidad de La Frontera (UFRO), Chile
Home Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Acute Lymphoblastic Leukemia (ALL) is the most common cancer in children worldwide and is characterized by the overproduction of undifferentiated lymphoblasts in bone marrow. The treatment of first choice is the enzyme L-asparaginase (ASNase) isolated from Escherichia coli or Erwinia chrysanthemi. Its mechanism of action is to hydrolyse plasma L-asparagine to aspartic acid and ammonia. Leukemic cells, unlike healthy cells, are unable to synthesize enough L-asparagine for their survival, due to a reduction in expression of the asparagine synthetase gene. These cells must rely on plasma L-asparagine for survival and hence, ASNase causes malignant cell death by starvation. ASNase formulations from E. coli and E. chrysanthemi can cause significant adverse side-effects, especially immunogenicity. PEGylation of E. coli ASNase is a post-translational modification strategy to minimize immunogenicity. PEGylatioon involves covalent attachment of polyethylene glycol (PEG) to camouflage epitopes on the enzyme. However, conformational restrictions imposed by covalent binding of PEG decreases enzyme activity and the immune system also raises antibodies against PEG. This project has the following goals: 1) to optimize culture conditions to produce a chimeric E. coli ASNase in a 3L bioreactor; 2) to purify and N-terminal PEGylate this ASNase; 3) to characterize the structure and function of the enzyme; 4) evaluate in vitro cytotoxicity of the enzyme against different leukaemic cell lines; and 5) evaluate in vivo asparagine and glutamine depletion, and immunogenicity of the PEGylated-chimeric enzyme in mouse models of ALL. These variants are expected to be active, physicochemically stable and less immunogenic than existing E. coli ASNase, providing a platform for future biopharmaceutical development. (AU)

Articles published in other media outlets (5 total):
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El Austral de Temuco online (Chile): Ufro y Fapesp trabajarán en la producción de enzima para tratar leucemia linfoblástica aguda por dos años (30/May/2021)
Prensa Ciudadana (Chile): Académicos de la UFRO y FAPESP de Brasil trabajarán en la producción de enzima para el tratamiento de la leucemia linfoblástica aguda (18/May/2021)
Temuco Univerciudad (Chile): Académicos de la UFRO y FAPESP de Brasil trabajarán en la producción de enzima para el tratamiento de la leucemia linfoblástica aguda (18/May/2021)
soychile.cl (Chile): Académicos de la Ufro y Fapesp de Brasil trabajarán en la producción de enzima para el tratamiento de la leucemia (18/May/2021)
UFRO - Universidad de La Frontera (Chile): Proyecto UFRO-FAPESP trabajará en la producción de enzima para el tratamiento de la leucemia linfoblástica aguda (12/May/2021)