| dc.description.abstracteng | The goal of the present work was to form a basis for the development of improved protein therapeutics against acute lymphoblastic leukemia (ALL). Bacterial L-asparaginases, in combination with other chemotherapeutics, are currently used for the treatment of ALL. This chemotherapy approach is limited by the elicitation of many immune responses to patients, mainly attributed to the bacterial origins of the used enzymes. A potential strategy to circumvent such imitations involves the replacement of the bacterial enzymes by human molecules which could drastically eliminate severe side effects arising from the immunogenicity. However, human enzymes which display L-asparaginase activity cannot be used for such treatment, because of their poor catalytic properties and therefore, protein engineering approaches for their catalytic improvement are inevitable. Overall, two novel human L-asparaginases were studied, namely human ASNase1 (hASNase1) and human ASNase3 (hASNase3), with the latter one being also structurally characterized.
Wildtype hASNase3 was used as template for mutagenesis and subsequent screening steps aiming at the identification of catalytically improved variants. A FACS-based high- throughput screening system was employed, which correlates semi-quantitatively the intracellular eGFP fluorescence intensity with the L-asparaginase activity. The system is based on a five-gene-deletion Escherichia coli (E.coli) strain (all genes which contribute to the biosynthesis of L-aspartate have been deleted) whose growth is exclusively dependent on the availability of exogenous L-aspartate, product of the L-asparaginase catalytic activity. The intracellular expression of hASNase3 variants can rescue the bacterial cells from the lack of L-aspartate since they can produce this amino acid through activity of these variants. The availability of L-aspartate reflects the expression levels of eGFP, and this, in turn, correlates the intracellular fluorescence with the L-asparaginase activity. Applying this screening strategy, overall five mutant libraries were analyzed (one generated via epPCR, and four via site-saturation mutagenesis), and eventually three human ASNase3 variants with improved catalytic properties against the hydrolysis of L-asparagine were identified and isolated, with the best one being 6-fold better than the wild type.
In addition, a novel high-throughput screening platform was developed by capitalizing on the rising field of droplet-based microfluidics. This approach allows the compartmentalization in very small water-in-oil emulsions of different types of chemical and/or enzymatic reactions, thereby monitoring the course of the reactions continuously. To this end, a novel fluorescent, three-step coupled assay for L-asparaginase was developed in order to be able to measure quantitatively enzymatic reactions in volumes of the range 500-600 pL. For standardizing the system at the single-cell level, the current antileukemic drug Escherichia coli L-asparaginase 2 was used, which was displayed on the inner membrane of E.coli cells. Individual cells displaying the enzyme were compartmentalized, and the assay was validated by measuring the activity of the displayed L-asparaginase. Our experimental results demonstrate that this setup allows the quantitative determination of single-cell enzymatic activities, thus being suitable for the screening of directed evolution mutant libraries not only for human L- asparaginases but also for other enzymes in general.
Besides hASNase3, we additionally characterized another human L-asparaginase, namely hASNase1. It was shown that this enzyme which comprises the N-terminal domain of an overall 60-kDa lysophospholipase and resembles the cytoplasmic bacterial E.coli L- asparaginase 1, can form an independent folding and catalytic unit. Strikingly, despite its monomeric state, hASNase1 displayed a very pronounced sigmoidal steady-state kinetic profile, hallmark of allosteric enzymes. Its catalytic properties are poorer than those of hASNase3, thus making its engineering task more challenging.
As a complementary strategy to the engineering of human enzymes for improvement of ALL therapy, we focused on the utilization of drug delivery approaches as means for the prolongation of the half-life of L-asparaginases under physiologically relevant conditions. By encapsulating Saccharomyces cerevisiae L-asparaginase 1 (ScASNase1) in multilayer polyelectrolyte microcapsules consisting of biocompatible and biodegradable materials, it was shown that the enzyme’s thermal stability and its resistance against proteolysis can be dramatically improved. In addition, the isothermal inactivation rate at 37 !C of the encapsulated enzyme was considerably lower as compared to the free enzyme, thus suggesting that the encapsulated enzyme can retain its activity at physiologically relevant temperatures longer than its free state. Ultimately, it was demonstrated that unlike preparations of free enzyme, microcapsules filled with active ScASNase1 can kill leukemic cells in-vitro even in the presence of a mixture of proteases which degrade the free enzyme. These results further suggest that encapsulation of the enzymes can prevent their degradation from proteases, thereby prolonging their half-life and consequently allowing them to kill
leukemic cells. Similar results were obtained when the experiments were done using E.coli L- asparaginase 2, the current antileukemic drug. | de |