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Made available in DSpace on 2021-06-25T10:35:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Ministério da Ciência, Tecnologia, Inovações e Comunicações Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs. Departamento de Tecnologia Bioquímico-Farmacêutica Faculdade de Ciências Farmacêuticas Universidade de São Paulo Centro Infantil Boldrini, Campinas Department of Medical Genetics Faculty of Medical Sciences State University of Campinas, Campinas Heart Institute (InCor) Medical School University of São Paulo Biosciences Institute UNESP – São Paulo State University Coastal Campus, São Vicente Department of Parasitology Biomedical Sciences Institute University of São Paulo Department of Clinical and Toxicological Analysis Faculdade de Ciências Farmacêuticas Universidade de São Paulo Biosciences Institute UNESP – São Paulo State University Coastal Campus, São Vicente FAPESP: 2013/08139-8 FAPESP: 2013/08617-7 FAPESP: 2014/06863-3 FAPESP: 2015/07749-2 FAPESP: 2016/25896-5 FAPESP: 2018/15104-0 FAPESP: 2018/15549-1 FAPESP: 2018/18257-1 CNPq: 28/2018 CNPq: 301596/2017-4 CAPES: 309595/2016-9 CNPq: 423532/2018-9