Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8 lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

The present study focuses on the formation of microcapsules containing catalytically active L-asparaginase (LASNase), a protein drug of high value in antileukemic therapy. We make use of the layer-by-layer (LbL) technique to coat protein-loaded calcium carbonate (CaCO 3 ) particles with two or three poly dextran/poly-L-arginine-based bilayers. To achieve high loading efficiency, the CaCO 3 template was generated by coprecipitation with the enzyme. After assembly of the polymer shell, the CaCO 3 core material was dissolved under mild conditions by dialysis against 20 mM EDTA. Biochemical stability of the encapsulated L-asparaginase was analyzed by treating the capsules with the proteases trypsin and thrombin, which are known to degrade and inactivate the enzyme during leukemia treatment, allowing us to test for resistance against proteolysis by physiologically relevant proteases through measurement of residual L-asparaginase activities. In addition, the thermal stability, the stability at the physiological temperature, and the longterm storage stability of the encapsulated enzyme were investigated. We show that encapsulation of L-asparaginase remarkably improves both proteolytic resistance and thermal inactivation at 37°C, which could considerably prolong the enzyme's in vivo half-life during application in acute lymphoblastic leukemia (ALL). Importantly, the use of low EDTA concentrations for the dissolution of CaCO 3 by dialysis could be a general approach in cases where the activity of sensitive biomacromolecules is inhibited, or even irreversibly damaged, when standard protocols for fabrication of such LbL microcapsules are used. Encapsulated and free enzyme showed similar efficacies in driving leukemic cells to apoptosis.

Human asparaginase 3 (hASNase3), which belongs to the N-terminal nucleophile (Ntn) hydrolase superfamily, is synthesized as a single polypeptide that is devoid of asparaginase activity. Intramolecular autoproteolytic processing releases the amino group of Thr168, a moiety required for catalyzing asparagine hydrolysis. Recombinant hASNase3 purifies as the uncleaved, asparaginase-inactive form, and undergoes self-cleavage to the active form at a very slow rate. Here we show that the free amino acid glycine selectively acts to accelerate hASNase3 cleavage both in vitro and in human cells. Other small amino acids such as alanine, serine, or the substrate asparagine are not capable of promoting autoproteolysis. Crystal structures of hASNase3 in complex with glycine in the uncleaved and cleaved enzyme states reveal the mechanism of glycine-accelerated post-translational processing, and explain why no other amino acid can substitute for glycine.

Background:The 60-kDa human lysophospholipase comprises an N-terminal domain with predicted, yet uncharacterized L-asparaginase activity and a C-terminal ankyrin repeat-like domain. Results:The N-terminal domain, termed hASNase1, was identified as a functional structural unit possessing catalytic activity. Conclusion: hASNase1 is an allosterically regulated bacterial-type cytoplasmic L-asparaginase. Significance: Domains of multifunctional human proteins harbor homologs of prokaryotic enzymes displaying similar structural and kinetic features.

Immunogenicity is one of the most common complications occurring during therapy making use of protein drugs of nonhuman origin. A notable example of such a case is bacterial l-asparaginases (L-ASNases) used for the treatment of acute lymphoblastic leukemia (ALL). The replacement of the bacterial enzymes by human ones is thought to set the basis for a major improvement of antileukemic therapy. Recently, we solved the crystal structure of a human enzyme possessing L-ASNase activity, designated hASNase-3. This enzyme is expressed as an inactive precursor protein and post-translationally undergoes intramolecular processing leading to the generation of two subunits which remain noncovalently, yet tightly associated and constitute the catalytically active form of the enzyme. We discovered that this intramolecular processing can be drastically and selectively accelerated by the free amino acid glycine. In the present study, we report on the molecular engineering of hASNase-3 aiming at the improvement of its catalytic properties. We created a fluorescence-activated cell sorting (FACS)-based high-throughput screening system for the characterization of rationally designed mutant libraries, capitalizing on the finding that free glycine promotes autoproteolytic cleavage, which activates the mutant proteins expressed in an E. coli strain devoid of aspartate biosynthesis. Successive screening rounds led to the isolation of catalytically improved variants showing up to 6-fold better catalytic efficiency as compared to the wild-type enzyme. Our work establishes a powerful strategy for further exploitation of the human asparaginase sequence space to facilitate the identification of in vitro-evolved enzyme species that will lay the basis for improved ALL therapy.

Leukemia L-Aspartate oxidase Coupled enzyme assay Amplex Red a b s t r a c tWe report on the development of a sensitive real-time assay for monitoring the activity of L-asparaginase that hydrolyzes L-asparagine to L-aspartate and ammonia. In this method, L-aspartate is oxidized by Laspartate oxidase to iminoaspartate and hydrogen peroxide (H 2 O 2 ), and in the detection step horseradish peroxidase uses H 2 O 2 to convert the colorless, nonfluorescent reagent Amplex Red to the red-colored and highly fluorescent product resorufin. The assay was validated in both the absorbance and the fluorescence modes. We show that, due to its high sensitivity and substrate selectivity, this assay can be used to measure enzymatic activity in human serum containing L-asparaginase.Ó 2013 Elsevier Inc. All rights reserved.The enzyme L-asparaginase (EC 3.5.1.1, L-asparagine amidohydrolase, L-ASNase), 1 which predominantly occurs in microorganisms and plants, catalyzes the hydrolysis of L-asparagine (L-Asn) to L-aspartic acid (L-Asp) and ammonia [1,2]. Escherichia coli L-ASNase has been used extensively as a therapeutic enzyme in the frontline treatment of lymphoblastic malignancies, such as acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma [3,4], since the 1960s. In light of the significance of L-ASNase as a therapeutic protein, various methods have been developed for measuring the enzyme's activity. Those assays can be used in either absorbance mode [5][6][7][8][9][10] or fluorescence mode [11,12]. However, these assays suffer from certain disadvantages that are primarily related to the use of substrate analogs instead of the natural substrate L-Asn [6,7,11,12]. Such assays are not suitable for in vitro evolution of L-asparaginases that aims to select variants showing improved catalytic efficiency and selectivity for the physiological substrate. Moreover, the limited sensitivity of absorption-based spectrophotometric assays [5][6][7]9,10] is a major handicap to significantly reducing reaction volumes. A noteworthy example of such a case is droplet-based microfluidics, which has emerged as a powerful tool for high-throughput screening in directed protein evolution [13,14]. In these experimental setups, the assay volume is minimized to 1 nl, to 1 fl, scaling down light path lengths to 1 lm.Here, we report on the development of a novel L-ASNase assay that can be used in either the fluorescence or absorbance mode and relies solely on the use of the physiological substrate L-Asn. In this three-step coupled enzyme system (Fig. 1), L-Asp, which is one of the two products of the L-ASNase reaction, is oxidized by L-aspartate oxidase (L-AspOx), resulting in the formation of iminoaspartate and hydrogen peroxide (H 2 O 2 ); the latter product is used by horseradish peroxidase (HRP) to oxidize the nonfluorescent compound Amplex Red (AR) to resorufin, which exhibits excellent fluorescence as well as absorbance properties.For establishing and quantitatively evaluating the assay, we cloned and recombinantly produced E. coli L-AspOx [15]...

We demonstrate the use of a hybrid microfluidic-micro-optical system for the screening of enzymatic activity at the single cell level. Escherichia coli b-galactosidase activity is revealed by a fluorogenic assay in 100 pl droplets. Individual droplets containing cells are screened by measuring their fluorescence signal using a high-speed camera. The measurement is parallelized over 100 channels equipped with microlenses and analyzed by image processing. A reinjection rate of 1 ml of emulsion per minute was reached corresponding to more than 10 5 droplets per second, an analytical throughput larger than those obtained using flow cytometry. V

Functional screenings in droplet-based microfluidics require the analysis of various types of activities of individual cells. When screening for enzymatic activities, the link between the enzyme of interest and the information-baring molecule, the DNA, must be maintained to relate phenotypes to genotypes. This linkage is crucial in directed evolution experiments or for the screening of natural diversity. Micro-organisms are classically used to express enzymes from nucleic acid sequences. However, little information is available regarding the most suitable expression system for the sensitive detection of enzymatic activity at the singlecell level in droplet-based microfluidics. Here, we compare three different expression systems for L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1), an enzyme of therapeutic interest that catalyzes the conversion of L-asparagine to L-aspartic acid and ammonia. We developed three expression vectors to produce and localize L-asparaginase (L-ASNase) in E. coli either in the cytoplasm, on the surface of the inner membrane (display), or in the periplasm. We show that the periplasmic expression is the most optimal strategy combining both a good yield and a good accessibility for the substrate without the need for lysing the cells. We suggest that periplasmic expression may provide a very efficient platform for screening applications at the single-cell level in microfluidics.