Preclinical in vivo assays to evaluate asparaginase biobetters

Abstract

L-asparagine (Asn) restriction has been an efficient strategy for the therapy of several cancers, especially hematological ones. Clinically, the best-known example of performing this restriction is the application of the enzyme L-asparaginase, responsible for hydrolyzing Asn into aspartic acid (Asp) and ammonia and, secondarily, glutamine (Gln) into glutamic acid (Glu) and ammonia. Recent data show the relationship between Asn depletion and inhibition of metastatic processes, which could greatly expand the use of ASNase for the treatment of metastatic solid tumors. However, in adults, ASNase is poorly tolerated by the immune system because it is an enzyme of bacterial origin. Thus, covering epitopes present in ASNase could be a good strategy to overcome immunogenicity, increasing the possibility of its wider clinical application. In previous projects funded by FAPESP (2013/08617-7 - thematic; 2015/07749-2 - regular and 2018/15104-0 - regular) our research group developed several improved versions (biobetters) of this enzyme, as well as evaluated pre-clinical parameters of some of them. In the last FAPESP grant, the preclinical study of protease-resistant and glycosylated proteoforms was proposed. However, due to the COVID-19 pandemic, only the study of protease-resistant proteoforms was completed. In this project, the objective is to carry out pre-clinical studies of the glycosylated proteoform of erwinase (Glicoerwi), as well as perform an in vivo comparison of the Glicoerwi with Erwinase produced in Escherichia coli and its pegylated version. Since the E. coli enzyme loses its activity when it is glycosylated, another proposal in this project will be the pre-clinical evaluation of E. coli asparaginase in complex with human ±2-macroglobulin. ±2-Macroglobulins (±2Ms) are among the largest and most relevant blood proteolytic inhibitors due to their universal ability to neutralize a broad spectrum of endopeptidases. Some studies have pointed to the possibility of ±2M encapsulating/trapping non-proteolytic proteins and enzymes and, once trapped, these molecules would be protected from the recognition of antibodies, human proteases and other blood components. Thus, human ±2-Macroglobulin can function as a unique medicinal vehicle/carrier that can be adapted for clinical use to improve ASNase tolerance. (AU)