Preclinical assays of glycoprotein asparaginase proteoforms or resistant to serum proteases.

Abstract

Acute Lymphoblastic Leukemia (ALL) is the cancer with the highest incidence in the age group of 3 to 5 years. Therapy for the treatment of ALL utilizes, among other drugs, the bacterial L-asparaginase enzyme of Escherichia coli (Ec_ASNase) and as a second option that of D. chrysanthemi (Er_ASNase). Immunogenic reactions are the major side effects related to ASNase use, and the formation of anti-ASNase antibodies makes treatment difficult. Two lysosomal cysteine proteases are involved to the degradation of Ec_ASNase in the bloodstream: Cathepsin B (CTSB) and asparaginyl endopeptidase (AEP). In previous studies in our laboratory, we obtained three Ec_ASNase mutants resistant to degradation by AEP and / or CTSB. In another front, we obtained asparaginases of E. coli and D. chrysanthemi expressed in Pichia pastoris, resulting in glycosylated proteoforms. All these recombinant proteins were the result of the thematic project FAPESP 2013 / 08617-7 (CIBio authorization attached), as well as of the Regular Project FAPESP 2015 / 07749-2. In this work, we propose to assess the cytotoxicity profile of these proteoforms by thiazolyl blue tetrazolium (MTT) and perform in vivo assays (CEUA authorization - FCF 560 attached) in Balb / C mice for evaluation of antibody formation and half-life of proteoforms in comparison with the wild-type enzymes expressed in E. coli bacteria (conventional system for obtaining L_ASNases). (AU)