A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

Trigalacturonate-producing pectate lyase PelQ1 from Saccharobesus litoralis with unique exolytic activity.

PelQ1 from Saccharobesus litoralis is a Ca2+-dependent pectate lyase belonging to the polysaccharide lyase family 1 (PL1). Although being an endolytic enzyme, it degraded polygalacturonate into predominantly unsaturated trimer in an exolytic manner with delayed production of dimer, tetramer and pentamer. The enzyme harbours a C-terminal domain from the carbohydrate-binding module family 13 (CBM13), whose presence facilitated the production of dimer. PelQ1's homology model showed that it possessed a well-conserved catalytic cleft, with R232 acting as the general base and R203 as the general acid. Structural comparison with DcPelC, a similar trimer-generating pectate lyase from Dickeya chrysanthemi EC16, implied that both enzymes' catalytic clefts encompassed at least eight subsites, i.e. -5 to +3. The unequal distribution of the subsites between the reducing and non-reducing ends of the cleavage site might be responsible for the exolytic generation of the trimer. As all but the -1, +1 and + 2 subsites could accommodate methylated galacturonate, this subclass of PL1 pectate lyases may function to help break up methylated pectin.

Development of a bioengineered Erwinia chrysanthemi asparaginase to enhance its anti-solid tumor potential for treating gastric cancer.

Asparaginase has been traditionally applied for only treating acute lymphoblastic leukemia due to its ability to deplete asparagine. However, its ultimate anticancer potential for treating solid tumors has not yet been unleashed. In this study, we bioengineered Erwinia chrysanthemi asparaginase (ErWT), one of the US Food and Drug Administration-approved types of amino acid depleting enzymes, to achieve double amino acid depletions for treating a solid tumor. We constructed a fusion protein by joining an albumin binding domain (ABD) to ErWT via a linker (GGGGS)5 to achieve ABD-ErS5. The ABD could bind to serum albumin to form an albumin-ABD-ErS5 complex, which could avoid renal clearance and escape from anti-drug antibodies, resulting in a remarkably prolonged elimination half-life of ABD-ErS5. Meanwhile, ABD-ErS5 did not only deplete asparagine but also glutamine for ∼2 weeks. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed tumor growth in an MKN-45 gastric cancer xenograft model, demonstrating a novel approach for treating solid tumor depleting asparagine and glutamine. Multiple administrations of ABD-ErS5 did not cause any noticeable histopathological abnormalities of key organs, suggesting the absence of acute toxicity to mice. Our results suggest ABD-ErS5 is a potential therapeutic candidate for treating gastric cancer.

Back to the future: the amazing journey of the therapeutic anti-leukemia enzyme asparaginase Erwinia chrysanthemi.

For several decades, asparaginase has been considered world-wide as an essential component of combination chemotherapy for the treatment of childhood acute lymphoblastic leukemia (ALL). Discovered over 60 years ago, two main unmanipulated asparaginase products originated from primary bacteria sources, namely Escherichia coli and Erwinia chrysanthemi, have been available for clinical use. A pegylated product of the Escherichia coli asparaginase was subsequently developed and is now the main product used by several international co-operative groups. The various asparaginase products all display the same mechanism of action (hydrolysis of circulating asparagine) and are associated with similar efficacy and toxicity patterns. However, their different pharmacokinetics, pharmacodynamics and immunological properties require distinctive modalities of application and monitoring. Erwinia chrysanthemi asparaginase was initially used as a first-line product, but subsequently became a preferred second-line product for children who experienced immunological reactions to the Escherichia coli asparaginase products. An asparaginase product displaying the same characteristics of the Erwinia chrysanthemi asparaginase has recently been produced by use of recombinant technology, thus securing a preparation available for use as an alternative, or as a back-up in case of shortages, for the non-recombinant product. The long journey of the Erwinia chrysanthemi asparaginase product as it has developed throughout the last several decades has made it possible for almost every child and adult with ALL to complete the asparaginase-based protocol treatment when an immunological reaction has occurred to any Escherichia coli asparaginase product.

Quality comparison of a state-of-the-art preparation of a recombinant L-asparaginase derived from Escherichia coli with an alternative asparaginase product.

L-asparaginase (ASNase) is a protein that is essential for the treatment of acute lymphoblastic leukemia (ALL). The main types of ASNase that are clinically used involve native and pegylated Escherichia coli (E. coli)-derived ASNase as well as Erwinia chrysanthemi-derived ASNase. Additionally, a new recombinant E. coli-derived ASNase formulation has received EMA market approval in 2016. In recent years, pegylated ASNase has been preferentially used in high-income countries, which decreased the demand for non-pegylated ASNase. Nevertheless, due to the high cost of pegylated ASNase, non-pegylated ASNase is still widely used in ALL treatment in low- and middle-income countries. As a consequence, the production of ASNase products from low- and middle-income countries increased in order to satisfy the demand worldwide. However, concerns over the quality and efficacy of these products were raised due to less stringent regulatory requirements. In the present study, we compared a recombinant E. coli-derived ASNase marketed in Europe (Spectrila®) with an E. coli-derived ASNase preparation from India (Onconase) marketed in Eastern European countries. To assess the quality attributes of both ASNases, an in-depth characterization was conducted. Enzymatic activity testing revealed a nominal enzymatic activity of almost 100% for Spectrila®, whereas the enzymatic activity for Onconase was only 70%. Spectrila® also showed excellent purity as analyzed by reversed-phase high-pressure liquid chromatography, size exclusion chromatography and capillary zone electrophoresis. Furthermore, levels of process-related impurities were very low for Spectrila®. In comparison, the E. coli DNA content in the Onconase samples was almost 12-fold higher and the content of host cell protein was more than 300-fold higher in the Onconase samples. Our results reveal that Spectrila® met all of the testing parameters, stood out for its excellent quality and, thus, represents a safe treatment option in ALL. These findings are particularly important for low- and middle-income countries, where access to ASNase formulations is limited.

Population pharmacokinetics of intramuscular recombinant Erwinia chrysanthemi asparaginase (JZP458) in patients with acute lymphoblastic leukemia.

JZP458 is a recombinant Erwinia chrysanthemi asparaginase for patients with acute lymphoblastic leukemia (ALL)/lymphoblastic lymphoma (LBL) who have developed hypersensitivity to Escherichia coli-derived asparaginases. A population pharmacokinetic (PopPK) model was developed for intramuscular (i.m.) JZP458 using serum asparaginase activity (SAA) data from 166 patients with ALL/LBL enrolled in a phase II/III study conducted in collaboration with the Children's Oncology Group (AALL1931; NCT04145531). The pharmacokinetics of i.m. JZP458 is best characterized by a one-compartment model with mixed-order absorption and linear elimination, with body surface area included as an allometric covariate on JZP458 SAA clearance and volume, and race (i.e., Black/African American) and disease subtype (i.e., T-cell ALL) as covariates on JZP458 SAA clearance. The PopPK model was used to simulate SAA profiles to estimate the likelihood of achieving nadir SAA (NSAA) levels greater than or equal to 0.1 IU/mL with different dosing regimens. Model-based simulations suggest when JZP458 is administered i.m. at 25/25/50 mg/m2 Monday/Wednesday/Friday (MWF), 92.1% of subjects (95% confidence interval [CI]: 90.9%, 93.3%) are expected to achieve the last 72-h (after 50 mg/m2 dose) NSAA level greater than or equal to 0.1 IU/mL, and 93.8% (95% CI: 92.7%, 94.9%) are expected to achieve the last 48-h (after 25 mg/m2 dose) NSAA level greater than or equal to 0.1 IU/mL. When JZP458 is administered 25 mg/m2 i.m. every 48 h, 93.8% (95% CI: 92.7%, 94.8%) are expected to achieve the last 48-h NSAA level greater than or equal to 0.1 IU/mL. These data supported the i.m. dose of 25 mg/m2 every 48 h or 25/25/50 mg/m2 on a MWF dosing schedule in patients with ALL/LBL.

Asparaginase therapy in patients with acute lymphoblastic leukemia: expert opinion on use and toxicity management.

The addition of asparaginase to acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL) treatment regimens provides significant patient benefits. Asparaginase therapies vary in origin (Escherichia coli- or Erwinia-derived) and preparation (native or pegylated), conferring distinct pharmacokinetic and immunogenic profiles. Clinical hypersensitivity reactions (HSRs) are commonly reported in patients and range from localized erythema to systemic anaphylaxis. Due to its favorable pharmacokinetic profile and reduced immunogenicity compared to native E. coli preparations, pegaspargase is the first-line asparaginase therapeutic option. Switching to an Erwinia-derived asparaginase is recommended for patients who experience HSRs or antibody-mediated inactivation to achieve the significant clinical benefit observed in patients who complete asparaginase treatment. Previous global shortages of asparaginase Erwinia chrysanthemi necessitated conversion mitigation strategies such as premedication protocols, desensitization, and asparaginase activity level monitoring. Here, we discuss the efficacy, safety, pharmacokinetics, current use, and administration of asparaginase therapies for pediatric and adolescent patients with ALL/LBL.

A systematic review of recent trends in research on therapeutically significant L-asparaginase and acute lymphoblastic leukemia.

L-asparaginases are mostly obtained from bacterial sources for their application in the therapy and food industry. Bacterial L-asparaginases are employed in the treatment of Acute Lymphoblastic Leukemia (ALL) and its subtypes, a type of blood and bone marrow cancer that results in the overproduction of immature blood cells. It also plays a role in the food industry in reducing the acrylamide formed during baking, roasting, and frying starchy foods. This importance of the enzyme makes it to be of constant interest to the researchers to isolate novel sources. Presently L-asparaginases from E. coli native and PEGylated form, Dickeya chrysanthemi (Erwinia chrysanthemi) are in the treatment regime. In therapy, the intrinsic glutaminase activity of the enzyme is a major drawback as the patients in treatment experience side effects like fever, skin rashes, anaphylaxis, pancreatitis, steatosis in the liver, and many complications. Its significance in the food industry in mitigating acrylamide is also a major reason. Acrylamide, a potent carcinogen was formed when treating starchy foods at higher temperatures. Acrylamide content in food was analyzed and pre-treatment was considered a valuable option. Immobilization of the enzyme is an advancing and promising technique in the effective delivery of the enzyme than in free form. The concept of machine learning by employing the Artificial Network and Genetic Algorithm has paved the way to optimize the production of L-asparaginase from its sources. Gene-editing tools are gaining momentum in the study of several diseases and this review focuses on the CRISPR-Cas9 gene-editing tool in ALL.

Structural Aspects of E. coli Type II Asparaginase in Complex with Its Secondary Product L-Glutamate.

Bacterial L-asparaginases are amidohydrolases (EC 3.5.1.1) capable of deaminating L-asparagine and, with reduced efficiency, L-glutamine. Interest in the study of L-asparaginases is driven by their use as biodrugs for the treatment of acute lymphoblastic leukemia. Here, we report for the first time the description of the molecular structure of type II asparaginase from Escherichia coli in complex with its secondary product, L-glutamate. To obtain high-quality crystals, we took advantage of the N24S variant, which has structural and functional features similar to the wild-type enzyme, but improved stability, and which yields more ordered crystals. Analysis of the structure of the N24S-L-glutamate complex (N24S-GLU) and comparison with its apo and L-aspartate-bound form confirmed that the enzyme-reduced catalytic efficiency in the presence of L-glutamine is due to L-glutamine misfitting into the enzyme-binding pocket, which causes a local change in the catalytic center geometry. Moreover, a tight interaction between the two protomers that form the enzyme active site limits the capability of L-glutamine to fit into (and to exit from) the binding pocket of E. coli L-asparaginase, explaining why the enzyme has lower glutaminolytic activity compared to other enzymes of the same family, in particular the Erwinia chrysanthemi one.

Asparaginase Erwinia chrysanthemi for acute lymphoblastic leukemia and lymphoblastic lymphoma.

Acute lymphoblastic leukemia (ALL) is a neoplastic disease characterized by the malignant proliferation of lymphoid cells in the blood and bone marrow. It accounts for approximately 75% of childhood leukemia. Lymphoblastic lymphoma (LBL) is a type of non-Hodgkin's lymphoma characterized by rapid growth and highly aggressive characteristics that occurs most commonly in adolescents and young adults. Asparaginase is primarily used to treat patients with ALL or LBL. Because allergic reactions occur in patients treated with bacterial-derived asparaginase, it is important to develop an alternative asparaginase preparation for patients allergic to asparaginase. Recombinant asparaginase Erwinia chrysanthemi-rywn (JZP-458) is a recombinant Erwinia asparaginase that uses a novel Pseudomonas fluorescens expression platform in the production process. JZP-458 has the same amino acid sequence as E. chrysanthemi-derived asparaginase (ERW) and its in vitro activity is similar to that of ERW. JZP-458 is highly efficacious in patients allergic to asparaginase. Data from a phase I clinical trial indicated that following the intramuscular or intravenous administration of JZP-458 to volunteers, serum asparaginase activity ≥ 0.1 IU/mL was observed in 100% of the volunteers 72 hours after administration. In this review, we summarize the mechanism of action and the related research data obtained with JZP-458 for the treatment of ALL or LBL.

L-asparaginase from Dickeya chrysanthemi: expression, purification and cytotoxicity assessment.

Microbial L-asparaginases are aminohydrolases that hydrolyze L-asparagine to L-aspartate. They are used to treat acute lymphoblastic leukemia and Hodgkin's lymphomas and in food industries. Increasing demand for L-ASNases is therefore needed. In the current study, the recombinant L-ASNase from Dickeya chrysanthemi (DcL-ASNase) was cloned into pET28a (+) expression vector and expressed in Escherichia coli as a 6His-tagged fusion protein and purified using Ni2+ chelated Sepharose chromatography resin, yielding a highly purified enzyme. Kinetics analysis allowed the determination of its substrate specificity and the physicochemical parameters that affect enzyme activity. The enzyme showed operational stability at 37 °C and 45 °C. The immunogenicity of the purified DcL-ASNase was evaluated by measuring the IgG and IgM levels in rats after injection. The cytotoxicity DcL-ASNase in selected cancer cell lines and peripheral blood monocytes was determined. The results showed that the enzyme induces pleiotropic effects, including significant morphological changes and the formation of apoptotic bodies. No cytotoxic effects were observed in peripheral blood monocytes at the same concentrations. In addition, gene expression analysis by RT-PCR of apoptotic biomarkers (Bax, survivin, and Ki-67) allowed the study of the apoptotic mechanism induced by DcL-ASNase on THP-1 cells.

Improved pharmacokinetic and pharmacodynamic profile of a novel PEGylated native Erwinia chrysanthemi L-Asparaginase.

INTRODUCTION: Erwinase® (native Erwinia chrysanthemi L-Asparaginase (nErA)) is an approved second-line treatment for acute lymphoblastic leukaemia (ALL) in children and adolescents, who develop hypersensitivity or neutralising antibodies to E.coli derived L-Asparaginases (ASNases). However, nErA has a short in vivo half-life requiring frequent dosing schedules in patients. In this study, nErA was covalently conjugated to PEG molecules with the aim of extending its half-life in vivo. METHODS: Firstly, efficacy of this novel product PEG-nErA was investigated on human ALL cell lines (Jurkat, CCRF-CEM and CCRF-HSB2), in vitro. Secondly, its pharmacokinetic (PK) and pharmacodynamic (PD) characteristics were determined, in vivo (12 rats in each group). Results. It was found that the specific activity (U/mg of enzyme) and the kinetic constant (KM) of nErA remained unaltered post PEGylation. PEG-nErA was shown to have similar cytotoxicity to nErA (IC50: 0.06-0.17 U/mL) on human ALL cell lines, in vitro. Further, when compared to nErA, PEG-nErA showed a significantly improved half-life in vivo, which meant that L-Asparagine (Asn) levels in plasma remained depleted for up to 25 days with a four-fold lower dose (100 U/kg) compared with 72 h for nErA at 400 U/kg dose. CONCLUSION: Overall, this next generation product PEG-nErA (with improved PK and PD characteristics compared to nErA) would bring a significant advantage to the therapeutic needs of ALL patients and should be further explored in clinical trials.

The Right-Handed Parallel ß-Helix Topology of Erwinia chrysanthemi Pectin Methylesterase Is Intimately Associated with Both Sequential Folding and Resistance to High Pressure.

The complex topologies of large multi-domain globular proteins make the study of their folding and assembly particularly demanding. It is often characterized by complex kinetics and undesired side reactions, such as aggregation. The structural simplicity of tandem-repeat proteins, which are characterized by the repetition of a basic structural motif and are stabilized exclusively by sequentially localized contacts, has provided opportunities for dissecting their folding landscapes. In this study, we focus on the Erwinia chrysanthemi pectin methylesterase (342 residues), an all-ß pectinolytic enzyme with a right-handed parallel ß-helix structure. Chemicals and pressure were chosen as denaturants and a variety of optical techniques were used in conjunction with stopped-flow equipment to investigate the folding mechanism of the enzyme at 25 °C. Under equilibrium conditions, both chemical- and pressure-induced unfolding show two-state transitions, with average conformational stability (ΔG° = 35 ± 5 kJ·mol-1) but exceptionally high resistance to pressure (Pm = 800 ± 7 MPa). Stopped-flow kinetic experiments revealed a very rapid (τ < 1 ms) hydrophobic collapse accompanied by the formation of an extended secondary structure but did not reveal stable tertiary contacts. This is followed by three distinct cooperative phases and the significant population of two intermediate species. The kinetics followed by intrinsic fluorescence shows a lag phase, strongly indicating that these intermediates are productive species on a sequential folding pathway, for which we propose a plausible model. These combined data demonstrate that even a large repeat protein can fold in a highly cooperative manner.

Can recombinant technology address asparaginase Erwinia chrysanthemi shortages?

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Bacterial L-asparaginase has played an important role in ALL treatment for several decades; however, hypersensitivity reactions to Escherichia coli-derived asparaginases often preclude their use. Inability to receive asparaginase due to hypersensitivities is associated with poor patient outcomes. Erwinia chrysanthemi-derived asparaginase (ERW) is an effective, non-cross-reactive treatment option, but is limited in supply. Consequently, alternative asparaginase preparations are needed to ensure asparaginase availability for patients with hypersensitivities. Recombinant technology can potentially address this unmet need by programming cells to produce recombinant asparaginase. JZP-458, a recombinant Erwinia asparaginase derived from a novel Pseudomonas fluorescens expression platform with no immunologic cross-reactivity to E. coli-derived asparaginases, has the same primary amino acid sequence as ERW, with comparable activity based on in vitro measurements. The efficient manufacturing of JZP-458 would provide an additional asparaginase preparation for patients with hypersensitivities.

L-asparaginase production review: bioprocess design and biochemical characteristics.

In the past decades, the production of biopharmaceuticals has gained high interest due to its great sensitivity, specificity, and lower risk of negative effects to patients. Biopharmaceuticals are mostly therapeutic recombinant proteins produced through biotechnological processes. In this context, L-asparaginase (L-asparagine amidohydrolase, L-ASNase (E.C. 3.5.1.1)) is a therapeutic enzyme that has been abundantly studied by researchers due to its antineoplastic properties. As a biopharmaceutical, L-ASNase has been used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), and other lymphoid malignancies, in combination with other drugs. Besides its application as a biopharmaceutical, this enzyme is widely used in food processing industries as an acrylamide mitigation agent and as a biosensor for the detection of L-asparagine in physiological fluids at nano-levels. The great demand for L-ASNase is supplied by recombinant enzymes from Escherichia coli and Erwinia chrysanthemi. However, production processes are associated to low yields and proteins associated to immunogenicity problems, which leads to the search for a better enzyme source. Considering the L-ASNase pharmacological and food importance, this review provides an overview of the current biotechnological developments in L-ASNase production and biochemical characterization aiming to improve the knowledge about its production. KEY POINTS: • Microbial enzyme applications as biopharmaceutical and in food industry • Biosynthesis process: from the microorganism to bioreactor technology • Enzyme activity and kinetic properties: crucial for the final application.

Determination of extent of PEGylation using denaturing capillary isoelectric focussing.

Conjugated proteins and enzymes are often formed using N-hydroxysuccinimide (NHS) chemistry, which reacts with free primary amines resulting in a loss of charge and a reduction in isoelectric point (pI). Measurement of the extent of reaction of these conjugates is critical for biopharmaceutical developers. Due to this change in protein charge state, denaturing capillary isoelectric focussing (cIEF) offers a potentially straightforward and convenient approach for extent-of-reaction quantification. Here, we demonstrate the potential of this technique with poly(ethylene glycol) (PEG) conjugates of Erwinia chrysanthemil-asparaginase (ErA). Development of an appropriate sample preparation technique is critical to achieving reproducible cIEF electropherograms, particularly for denaturation-resistant proteins such as ErA, and an emphasis was placed on this during development of the PEG-ErA cIEF method. cIEF electropherograms demonstrating a distribution of PEGylation states in a bell-shaped curve were obtained, and assignment of PEGylation states to these peaks was critical to routine use of the method. The method is sensitive enough to resolve non-lysine adducts of PEG (such as those conjugated to histidine residues) and was shown to give reproducible results over a 2 year period. Biopharmaceutical developers should consider cIEF for extent of reaction monitoring and measurement for conjugates of free amine groups.

Mechanism of Catalysis by l-Asparaginase.

Two bacterial type II l-asparaginases, from Escherichia coli and Dickeya chrysanthemi, have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lymphomas. Despite a long history of successful pharmacological applications and the apparent simplicity of the catalytic reaction, controversies still exist regarding major steps of the mechanism. In this report, we provide a detailed description of the reaction catalyzed by E. coli type II l-asparaginase (EcAII). Our model was developed on the basis of new structural and biochemical experiments combined with previously published data. The proposed mechanism is supported by quantum chemistry calculations based on density functional theory. We provide strong evidence that EcAII catalyzes the reaction according to the double-displacement (ping-pong) mechanism, with formation of a covalent intermediate. Several steps of catalysis by EcAII are unique when compared to reactions catalyzed by other known hydrolytic enzymes. Here, the reaction is initiated by a weak nucleophile, threonine, without direct assistance of a general base, although a distant general base is identified. Furthermore, tetrahedral intermediates formed during the catalytic process are stabilized by a never previously described motif. Although the scheme of the catalytic mechanism was developed only on the basis of data obtained from EcAII and its variants, this novel mechanism of enzymatic hydrolysis could potentially apply to most (and possibly all) l-asparaginases.