Downstream process and evaluation of the concomitant impact of a recombinant glycosylated L-asparaginase on leukemic cancer cells and the bone marrow tumor microenvironment

Author(s)

Kleingesinds, Eduardo Krebs ; Parizotto, Leticia de Almeida ; Effer, Brian ; Monteiro, Gisele ; Long, Paul F. ; Arroyo-Berdugo, Yoana ; Behrends, Volker ; Esposito, Maria Teresa ; Calle, Yolanda ; Pessoa-Jr, Adalberto

• Document type: Journal article
• Source: Process Biochemistry; v. 131, p. 11-pg., 2023-06-09.

Abstract

L-asparaginase (L-ASNase) is a life-saving medication used in the treatment of Acute Lymphoblastic Leukemia (ALL) which affects over 60,000 people yearly. However, L-ASNase still requires improvement since up to 60% of the patients develop hypersensitivity due to its immunogenicity. To address this issue, this work describes the downstream process of an L-ASNase from Erwinia chrysanthemi expressed extracellularly by Pichia pastoris with human-like glycosylation pattern and its further impact on leukemic cells co-cultured with bone marrow (BM) cytoprotective stromal cells. After size exclusion chromathography, a final yield of 54.93% was achieved and the proteomics analyses confirmed the attainment of an extremely pure enzyme. Glycosylated L-ASNase induced the complete inhibition of the proliferation of ALL and Acute Myeloid Leukemia (AML) cell lines tested, independently of the presence of BM stromal cells. However, the cytotoxic efficacy (induction of apoptosis) of glycosylated L-ASNase varied across cell lines, with ALL cell lines showing the most sensitivity. Additionally, the proapoptotic effect of glycosylated L-ASNase was partially inhibited by BM stromal cells. Taken together, our data warrant further investigations for the use of glycosylated L-ASNase against ALL and AML that should take into consideration the mechanisms of resistance mediated by the BM stroma for improved efficacy. (AU)

• FAPESP's process: 19/06919-2 - In vivo test of humanized L-asparaginase free and nano-encapsulated in liposomes
• Grantee: Eduardo Krebs Kleingesinds
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• FAPESP's process: 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical
• Grantee: Adalberto Pessoa Junior
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 15/07749-2 - Protein engineering and comparison of microbial expression systems of the biopharmaceutical L-asparaginase
• Grantee: Gisele Monteiro
• Support Opportunities: Regular Research Grants
• FAPESP's process: 18/03734-9 - Implementation of Quality by Design to the Development of the Production Process of the Chemotherapeutical Enzyme L- Asparaginase
• Grantee: Francisco Vitor Santos da Silva
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 17/25065-9 - Production of L-asparaginase by Pichia pastoris in fed-batch bioreactors
• Grantee: Letícia de Almeida Parizotto
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 17/20384-9 - Development of downstream process of humanized L-asparaginase and its characterization
• Grantee: Eduardo Krebs Kleingesinds
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 18/15104-0 - Preclinical assays of glycoprotein asparaginase proteoforms or resistant to serum proteases.
• Grantee: Gisele Monteiro
• Support Opportunities: Regular Research Grants