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

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 pro-apoptotic 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.