| Grant number: | 20/06982-3 |
| Support Opportunities: | Regular Research Grants |
| Start date: | May 01, 2021 |
| End date: | April 30, 2023 |
| Field of knowledge: | Biological Sciences - Microbiology - Applied Microbiology |
| Agreement: | Universidad de la Frontera |
| Principal Investigator: | Adalberto Pessoa Junior |
| Grantee: | Adalberto Pessoa Junior |
| Principal researcher abroad: | Jorge Gonzalo Farias Avendaño |
| Institution abroad: | Universidad de La Frontera (UFRO), Chile |
| Host 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)
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