Asparaginase is commonly used in combination therapy of acute lymphoblastic leukemia. However, as an immunogenic protein, hypersensitivity reactions (HSRs) during asparaginase therapy are frequent, ...indicating the development of anti-asparaginase antibodies. These can be associated with diminished clinical effectiveness, including poorer survival. Therapeutic drug monitoring of serum asparaginase activity to confirm complete asparagine depletion is therefore crucial during asparaginase therapy. Switching to alternative types of asparaginase is recommended for patients experiencing HSRs or silent inactivation; those with HSRs or silent inactivation on
derived asparaginases should switch to another preparation. However, prior global shortages of
asparaginase highlight the importance of alternative non-
derived asparaginase, including recombinant
asparaginase.
Summary
Asparaginase is essential in treating acute lymphoblastic leukaemia (ALL). Asparaginase‐related hypersensitivity causes treatment discontinuation, which is associated with decreased ...event‐free survival. To continue asparaginase treatment after hypersensitivity, a formulation of asparaginase encapsulated in erythrocytes (eryaspase) was developed. In NOR‐GRASPALL 2016 (NCT03267030) the safety and efficacy of eryaspase was evaluated in 55 patients (aged 1–45 years; median: 6.1 years) with non‐high‐risk ALL and hypersensitivity to asparaginase conjugated with polyethylene glycol (PEG‐asparaginase). Eryaspase (150 u/kg) was scheduled to complete the intended course of asparaginase (1–7 doses) in two Nordic/Baltic treatment protocols. Forty‐nine (96.1%) patients had asparaginase enzyme activity (AEA) ≥100 iu/l 14 ± 2 days after the first eryaspase infusion median AEA 511 iu/l; interquartile range (IQR), 291–780, whereas six of nine (66.7%) patients had AEA ≥100 iu/l 14 ± 2 days after the fourth infusion (median AEA 932 iu/l; IQR, 496–163). The mean terminal half‐life of eryaspase following the first infusion was 15.3 ± 15.5 days. Few asparaginase‐related adverse events were reported; five patients (9.1%) developed clinical allergy associated with enzyme inactivation. Replacement therapy was successfully completed in 50 patients (90.9%). Eryaspase was well tolerated, and most patients had AEA levels above the therapeutic target after the first infusion. The half‐life of eryaspase confirmed that a 2‐week schedule is appropriate.
L-asparaginase (ASNase) from Escherichia coli is currently used in some countries in its PEGylated form (ONCASPAR, pegaspargase) to treat acute lymphoblastic leukemia (ALL). PEGylation refers to the ...covalent attachment of poly(ethylene) glycol to the protein drug and it not only reduces the immune system activation but also decreases degradation by plasmatic proteases. However, pegaspargase is randomly PEGylated and, consequently, with a high degree of polydispersity in its final formulation. In this work we developed a site-specific N-terminus PEGylation protocol for ASNase. The monoPEG-ASNase was purified by anionic followed by size exclusion chromatography to a final purity of 99%. The highest yield of monoPEG-ASNase of 42% was obtained by the protein reaction with methoxy polyethylene glycol-carboxymethyl N-hydroxysuccinimidyl ester (10kDa) in 100 mM PBS at pH 7.5 and PEG:ASNase ratio of 25:1. The monoPEG-ASNase was found to maintain enzymatic stability for more days than ASNase, also was resistant to the plasma proteases like asparaginyl endopeptidase and cathepsin B. Additionally, monoPEG-ASNase was found to be potent against leukemic cell lines (MOLT-4 and REH) in vitro like polyPEG-ASNase. monoPEG-ASNase demonstrates its potential as a novel option for ALL treatment, being an inventive novelty that maintains the benefits of the current enzyme and solves challenges.
l-asparaginase (ASNase, EC 3.5.1.1) is an aminohydrolase enzyme with important uses in the therapeutic/pharmaceutical and food industries. Its main applications are as an anticancer drug, mostly for ...acute lymphoblastic leukaemia (ALL) treatment, and in acrylamide reduction when starch-rich foods are cooked at temperatures above 100 °C. Its use as a biosensor for asparagine in both industries has also been reported. However, there are certain challenges associated with ASNase applications. Depending on the ASNase source, the major challenges of its pharmaceutical application are the hypersensitivity reactions that it causes in ALL patients and its short half-life and fast plasma clearance in the blood system by native proteases. In addition, ASNase is generally unstable and it is a thermolabile enzyme, which also hinders its application in the food sector. These drawbacks have been overcome by the ASNase confinement in different (nano)materials through distinct techniques, such as physical adsorption, covalent attachment and entrapment. Overall, this review describes the most recent strategies reported for ASNase confinement in numerous (nano)materials, highlighting its improved properties, especially specificity, half-life enhancement and thermal and operational stability improvement, allowing its reuse, increased proteolysis resistance and immunogenicity elimination. The most recent applications of confined ASNase in nanomaterials are reviewed for the first time, simultaneously providing prospects in the described fields of application.
We succeeded in homogeneously expressing and purifying
l-
asparaginase from
Latilactobacillus sakei
LK-145 (
Ls
-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, ...C264S/C290S, and C196S/C264S/C290S-
Ls
-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of
Ls
-Asn1 were found to affect the catalytic activity of
Ls
-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-
Ls
-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of
Ls
-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-
Ls
-Asn1) were investigated for comparison with
Ls
-Asn1. We found that only C196S-
Ls
-Asn1 has almost the same enzymatic properties as that of
Ls
-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl
2
, and FeCl
2
. We measured the growth inhibitory effect of
Ls
-Asn1 and C196S-
Ls
-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using
l
-asparaginase from
Escherichia coli
K-12 as a reference. Only C196S-
Ls
-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that
Ls
-Asn1 is a so-called “Type IIb” novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results,
Ls
-Asn1 is expected to be useful as a new leukemia therapeutic agent.
l
-asparaginase (LA) catalyzes the degradation of asparagine, an essential amino acid for leukemic cells, into ammonia and aspartate. Owing to its ability to inhibit protein biosynthesis in ...lymphoblasts, LA is used to treat acute lymphoblastic leukemia (ALL). Different isozymes of this enzyme have been isolated from a wide range of organisms, including plants and terrestrial and marine microorganisms. Pieces of information about the three-dimensional structure of
l
-asparaginase from
Escherichia coli
and
Erwinia
sp. have identified residues that are essential for catalytic activity. This review catalogues the major sources of
l
-asparaginase, the methods of its production through the solid state (SSF) and submerged (SmF) fermentation, purification, and characterization as well as its biological roles. In the same breath, this article explores both the past and present applications of this important enzyme and discusses its future prospects.
L-asparaginase having low glutaminase has been a key therapeutic agent in the treatment of acute lymphpoblastic leukemia (A.L.L). In the present study, an extracellular L-asparaginase with low ...glutaminase activity, produced by Bacillus licheniformis was purified to homogeneity. Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine. The activity of purified L-asparaginase enhanced in presence of cations including Na+ and K+, whereas it was moderately inhibited in the presence of divalent cations and thiol group blocking reagents. The purified enzyme was maximally active over the range of pH 6.0 to 10.0 and temperature of 40°C and enzyme was stable maximum at pH 9.0 and -20°C. CD spectra of L-asparaginase predicted the enzyme to consist of 63.05% α-helix and 3.29% β-sheets in its native form with T222 of 58°C. Fluorescent spectroscopy showed the protein to be stable even in the presence of more than 3 M GdHCl. Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10(-5) M, 4.03 IU and 2.68×10(3) s(-1), respectively. The purified L-asparaginase had cytotoxic activity against various cancerous cell lines viz. Jurkat clone E6-1, MCF-7 and K-562 with IC50 of 0.22 IU, 0.78 IU and 0.153 IU respectively. However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug.
l
-asparaginase is a critical part of the treatment of acute lymphoblastic leukaemia in children and adolescents, and has contributed to the improvement in patient outcomes over the last 40 years. ...The main products used in clinical treatment are
l
-asparaginase enzymes derived from
Escherichia coli
and
Erwinia chrysanthemi
. However, a very active area of research is the identification and characterisation of potential new
l
-asparaginase therapeutics, from existing or novel prokaryotic and eukaryotic sources, including mutations to improve function. In this review, we discuss the critical factors necessary to adequately characterise novel
l
-asparaginase therapeutic products, including enzyme kinetic parameters, glutaminase activity, and toxicity. One critical consideration is to ensure that the substrate affinity of novel enzymes, as measured by the Michaelis constant K
M
, is sufficiently low to enable efficient reaction rates in human clinical use. The activity of
l
-asparaginases towards glutamine as a substrate is discussed and reviewed in detail, as there is much debate in the scientific literature about the importance of this feature for therapeutic enzymes. The recent research in the area is reviewed, including identification of new sources of the enzyme, modulating glutaminase activity, and improving the thermal stability and immunogenic response. New research in the area may benefit from these considerations, to enable the next generation of therapeutic product design. Critical to future work in this area is a complete characterisation of novel enzymes with respect to performance for both
l
-asparagine and
l
-glutamine as substrates.
l‐Asparaginases hydrolyzing plasma l‐asparagine and l‐glutamine has attracted tremendous attention in recent years owing to remarkable anticancer properties. This enzyme is efficiently used for acute ...lymphoblastic leukemia (ALL) and lymphosarcoma and emerged against ALL in children, neoplasia, and some other malignancies. Cancer cells reduce the expression of l‐asparaginase leading to their elimination. The l‐asparaginase anticancerous application approach has made incredible breakthrough in the field of modern oncology through depletion of plasma l‐asparagine to inhibit the cancer cells growth; particularly among children. High level of l‐asparaginase enzyme production by Escherichia coli, Erwinia species, Streptomyces, and Bacillus subtilis species is highly desirable as bacterial alternative enzyme sources for anticancer therapy. Thermal or harsh conditions stability of those from the two latter bacterial species is considerable. Some enzymes from marine bacteria have conferred stability in adverse conditions being more advantageous in cancer therapy. Several side effects exerted by l‐asparaginases such as hypersensitivity should be hindered or decreased through alternative therapies or use of immune‐suppressor drugs. The l‐asparaginase from Erwinia species has displayed remarkable traits in children with this regard. Noticeably, Erwinia chrysanthemi l‐asparaginase exhibited negligible glutaminase activity representing a promising efficiency mitigating related side effects. Application of software such as RSM would optimize conditions for higher levels of enzyme production. Additionally, genetic recombination of the encoding gene would indisputably help improving enzyme traits. Furthermore, the possibility of anticancer combination therapy using two or more l‐asparaginases from various sources is plausible in future studies to achieve better therapeutic outcomes with lower side effects.
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