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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

The drug l-asparaginase is a cornerstone in the treatment of acute lymphoblastic leukemia (ALL). The native E. colil-asparaginase used in Brazil until recently has been manufactured by Medac/Kyowa. Then a decision was taken by the Ministry of Health in 2017 to supply the National Health System with a cheaper alternative l-asparaginase manufactured by Beijing SL Pharmaceutical, called Leuginase®. As opposed to Medac, the asparaginase that has been in use in Brazil under the trade name of Aginasa®, it was not possible to find a single entry with the terms Leuginase in the Pubmed repository. The apparent lack of clinical studies and the scarcity of safety information provided to the hospitals by the drug distributor created a debate among Brazilian pediatric oncologists about issues of safety and efficacy that culminated eventually in a court decision to halt the distribution of the new drug all over the country. Boldrini Children's Center, a non-profit pediatric oncohematology hospital, has conducted its own evaluation of Leuginase®. Mass spectrometry analyses found at least 12 different contaminating host-cell proteins (HCP) in Leuginase®. The presence of two HCP (beta-lactamase and malate dehydrogenase) was confirmed by orthogonal methodologies. The relative number of HCP peptides ranged from 19 to 37% of the total peptides identified by mass spectrometry. In vivo studies in mice injected with Leuginase® revealed a 3 times lower plasma bioavailability and the development of higher antibody titres against l-asparaginase in comparison to Aginasa®-injected animals. The decision to buy a new drug based on its price alone is not safe. Developing countries are especially vulnerable to cheaper alternatives that lack solid quality assurance., Highlights • l-asparaginase is one of the first biologicals to be used and an essential drug for the treatment of acute lymphoblastic leukemia (ALL). • Current access to technology has facilitated the worldwide production of l-asparaginase and other biosimilars at lower prices. • Different quality control standards among countries pose a serious challenge to warrant quality, safety and efficacy of biosimilars. l-asparaginase is a cornerstone drug in the treatment of acute lymphoblastic leukemia (ALL). A cheaper l-asparaginase biosimilar was introduced in the Brazilian market in 2017. This report describes the bioanalytical evaluation of such biosimilar, which revealed the presence of at least 12 different contaminating host-cell proteins (HCP), that accounted for 19 to 37% of the peptides identified by three independent mass spectrometry analyses. The second most abundant HCP alone was confirmed by an orthogonal method to represent 2.4% of the total protein mass. Studies carried out in mice showed lower plasma bioavailability and higher anti-asparaginase antibodies titres for the biosimilar in comparison to the well-known Medac l-asparaginase previously used in the Country.