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.

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.

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.

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.

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.

A comprehensive review on microbial l-asparaginase: Bioprocessing, characterization, and industrial applications.

l-Asparaginase (E.C.3.5.1.1.) is a vital enzyme that hydrolyzes l-asparagine to l-aspartic acid and ammonia. This property of l-asparaginase inhibits the protein synthesis in cancer cells, making l-asparaginase a mainstay of pediatric chemotherapy practices to treat acute lymphoblastic leukemia (ALL) patients. l-Asparaginase is also recognized as one of the important food processing agent. The removal of asparagine by l-asparaginase leads to the reduction of acrylamide formation in fried food items. l-Asparaginase is produced by various organisms including animals, plants, and microorganisms, however, only microorganisms that produce a substantial amount of this enzyme are of commercial significance. The commercial l-asparaginase for healthcare applications is chiefly derived from Escherichia coli and Erwinia chrysanthemi. A high rate of hypersensitivity and adverse reactions limits the long-term clinical use of l-asparaginase. Present review provides thorough information on microbial l-asparaginase bioprocess optimization including submerged fermentation and solid-state fermentation for l-asparaginase production, downstream purification, its characterization, and issues related to the clinical application including toxicity and hypersensitivity. Here, we have highlighted the bioprocess techniques that can produce improved and economically viable yields of l-asparaginase from promising microbial sources in the current scenario where there is an urgent need for alternate l-asparaginase with less adverse effects.

Acidic isoforms of Erwinase form part of the product: Correlation with clinical experience.

Erwinia chrysanthemil-asparaginase (ErA) has been used for the treatment of acute lymphoblastic leukaemia (ALL) for decades, and its safety and efficacy have been well demonstrated. ErA drug substance and drug product contain a small proportion of acidic isoforms, with a known mechanism of formation, which have been shown to be minor conformational variants retaining enzymatic activity and function. Specifications for these acidic isoforms were set with an extremely limited data set, and with further manufacturing experience, it can now be demonstrated that they were set too tightly. Here, we consider the ability of the manufacturing process to meet the current acidic isoforms specifications, as well as clinical outcomes from drug product containing a higher proportion of isoforms. Compared with the historical clinical experience with the drug, there appeared to be no difference in the rate of adverse event reporting (e.g., hypersensitivity or other events) when drug product with relatively higher acidic isoforms was administered. ErA acidic isoforms comprise part of the ErA product and appear to have no clinical relevance, so a realignment of process capability and specification may be warranted. Biopharmaceutical developers should exercise caution when setting specifications with limited data, to avoid process capability pitfalls later.

Decreasing the immunogenicity of Erwinia chrysanthemi asparaginase via protein engineering: computational approach.

Immunogenicity of therapeutic proteins is one of the main challenges in disease treatment. L-Asparaginase is an important enzyme in cancer treatment which sometimes leads to undesirable side effects such as immunogenic or allergic responses. Here, to decrease Erwinase (Erwinia chrysanthemiL-Asparaginase) immunogenicity, which is the main drawback of the enzyme, firstly conformational B cell epitopes of Erwinase were predicted from three-dimensional structure by three different computational methods. A few residues were defined as candidates for reducing immunogenicity of the protein by point mutation. In addition to immunogenicity and hydrophobicity, stability and binding energy of mutants were also analyzed computationally. In order to evaluate the stability of the best mutant, molecular dynamics simulation was performed. Among mutants, H240A and Q239A presented significant reduction in immunogenicity. In contrast, the immunogenicity scores of D235A slightly decreased according to two servers. Binding affinity of substrate to the active site reduced significantly in K265A and E268A. The final results of molecular dynamics simulation indicated that H240A mutation has not changed the stability, flexibility, and the total structure of desired protein. Overall, point mutation can be used for reducing immunogenicity of therapeutic proteins, in this context, in silico approaches can be used to screen suitable mutants.

PEGylated E. coli asparaginase desensitization: an effective and feasible option for pediatric patients with acute lymphoblastic leukemia who have developed hypersensitivity to pegaspargase in the absence of asparaginase Erwinia chrysanthemi availability.

Asparaginase is an important component of multi-agent chemotherapy for the treatment of pediatric acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LLy). Hypersensitivity to the PEGylated form, pegaspargase, is the most common toxicity observed and is ideally addressed by substituting multiple doses of erwinia asparaginase for each subsequent dose of pegaspargase. An international shortage of erwinia asparaginase has limited the therapeutic options for those experiencing pegaspargase hypersensitivity. Here, we report pegaspargase can be safely administered, while maintaining sustained levels of asparaginase activity, to patients who have had a prior hypersensitivity reaction to pegaspargase by using a standard rapid desensitization protocol. Ten patients with prior hypersensitivity reactions to pegaspargase were treated by using a standardized rapid desensitization protocol. Eight patients had therapeutic asparaginase levels between days 4 and 7 of ≥0.05 IU/mL, and seven patients continued to have sustained levels above ≥0.1 IU/mL between days 10 and 14. Based on chemotherapy regimens, five of these patients successfully received more than one dose of pegaspargase utilizing this protocol.

L-Asparaginase from E. chrysanthemi expressed in glycoswitch®: effect of His-Tag fusion on the extracellular expression.

L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.

Symptomatic Hyperammonemia With Erwinia chrysanthemi-derived Asparaginase in Pediatric Leukemia Patients.

Erwinia chrysanthemi-derived asparaginase is increasingly integral to acute lymphoblastic leukemia therapy. In our series, 16% of patients developed symptomatic hyperammonemia following Erwinia administration with symptoms including refractory nausea, vomiting, profound fatigue, malaise, and coma. This series of patients receiving Erwinia indicates higher than expected incidence of hyperammonemia, correlation between ammonia and asparaginase levels and therapeutic asparaginase activity levels despite dose reduction. The series provides evidence for investigation into which patients require intervention to prevent toxicity, which patients may have ammonia levels used as an asparaginase activity surrogate and which patients may achieve equivalent efficacy with abridged dosing.

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity.

Current FDA-approved l-asparaginases also possess significant l-glutaminase activity, which correlates with many of the toxic side effects of these drugs. Therefore, l-asparaginases with reduced l-glutaminase activity are predicted to be safer. We exploited our recently described structures of the Erwinia chrysanthemi l-asparaginase (ErA) to inform the design of mutants with diminished ability to hydrolyze l-glutamine. Structural analysis of these variants provides insight into the molecular basis for the increased l-asparagine specificity. A primary role is attributed to the E63Q mutation that acts to hinder the correct positioning of l-glutamine but not l-asparagine. The substitution of Ser-254 with either an asparagine or a glutamine increases the l-asparagine specificity but only when combined with the E63Q mutation. The A31I mutation reduces the substrate Km value; this is a key property to allow the required therapeutic l-asparagine depletion. Significantly, an ultra-low l-glutaminase ErA variant maintained its cell killing ability. By diminishing the l-glutaminase activity of these highly active l-asparaginases, our engineered ErA variants hold promise as l-asparaginases with fewer side effects.

Structural Insight into Substrate Selectivity of Erwinia chrysanthemi L-asparaginase.

l-Asparaginases of bacterial origin are a mainstay of acute lymphoblastic leukemia treatment. The mechanism of action of these enzyme drugs is associated with their capacity to deplete the amino acid l-asparagine from the blood. However, clinical use of bacterial l-asparaginases is complicated by their dual l-asparaginase and l-glutaminase activities. The latter, even though representing only ∼10% of the overall activity, is partially responsible for the observed toxic side effects. Hence, l-asparaginases devoid of l-glutaminase activity hold potential as safer drugs. Understanding the key determinants of l-asparaginase substrate specificity is a prerequisite step toward the development of enzyme variants with reduced toxicity. Here we present crystal structures of the Erwinia chrysanthemi l-asparaginase in complex with l-aspartic acid and with l-glutamic acid. These structures reveal two enzyme conformations-open and closed-corresponding to the inactive and active states, respectively. The binding of ligands induces the positioning of the catalytic Thr15 into its active conformation, which in turn allows for the ordering and closure of the flexible N-terminal loop. Notably, l-aspartic acid is more efficient than l-glutamic acid in inducing the active positioning of Thr15. Structural elements explaining the preference of the enzyme for l-asparagine over l-glutamine are discussed with guidance to the future development of more specific l-asparaginases.

Activity and Toxicity of Intravenous Erwinia Asparaginase Following Allergy to E. coli-Derived Asparaginase in Children and Adolescents With Acute Lymphoblastic Leukemia.

BACKGROUND: Erwinia asparaginase is antigenically distinct from E.coli-derived asparaginase and may be used after E.coli-derived asparaginase hypersensitivity. In a single-arm, multicenter study, we evaluated nadir serum asparaginase activity (NSAA) and toxicity with intravenously administered asparaginase Erwinia chrysanthemi (IV-Erwinia) in children and adolescents with acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma with hypersensitivity to E.coli-derived asparaginase. PATIENTS AND METHODS: Between 2012 and 2013, 30 patients (age 1-17 years) enrolled from 10 centers. Patients received IV-Erwinia, 25,000 IU/m(2)/dose on Monday/Wednesday/Friday, for 2 consecutive-weeks (6 doses = 1 cycle) for each dose of pegaspargase remaining in the original treatment plan. The primary objective was to determine the proportion of patients achieving NSAA ≥ 0.1 IU/ml 48 hr after dose 5 in Cycle 1. Secondary objectives included determining the proportion achieving NSAA ≥ 0.1 IU/ml 72 hr after Cycle 1 dose 6, and the frequency of asparaginase-related toxicities. RESULTS: Twenty-six patients completed Cycle 1; 24 were evaluable for NSAA assessment. In Cycle 1, NSAA ≥ 0.10 IU/ml was detected in 83% of patients (95% confidence interval [CI], 63-95%) 48 hr post-dose 5 (mean ± SD; 0.32 IU/ml ± 0.23), and in 43% (95% CI, 22-66%) 72 hr post-dose 6 (mean ± SD; 0.089 IU/ml ± 0.072). For all 30 patients over all cycles, hypersensitivity/infusional reactions with IV-Erwinia occurred in 37%, pancreatitis 7%, and thrombosis 3%. CONCLUSIONS: IV-Erwinia administration in children/adolescents appeared feasible and tolerable. A therapeutically-effective NSAA (≥ 0.10 IU/ml) was achieved in most patients at 48 hr, but in fewer than half 72 hr post-dosing, suggesting that monitoring NSAA levels and/or every 48 hr dosing may be indicated.

"Superchiral" Spectroscopy: Detection of Protein Higher Order Hierarchical Structure with Chiral Plasmonic Nanostructures.

Optical spectroscopic methods do not routinely provide information on higher order hierarchical structure (tertiary/quaternary) of biological macromolecules and assemblies. This necessitates the use of time-consuming and material intensive techniques, such as protein crystallography, NMR, and electron microscopy. Here we demonstrate a spectroscopic phenomenon, superchiral polarimetry, which can rapidly characterize ligand-induced changes in protein higher order (tertiary/quaternary) structure at the picogram level, which is undetectable using conventional CD spectroscopy. This is achieved by utilizing the enhanced sensitivity of superchiral evanescent fields to mesoscale chiral structure.

Allergic reactions and antiasparaginase antibodies in children with high-risk acute lymphoblastic leukemia: A children's oncology group report.

BACKGROUND: The objectives of this study were to assess the incidence of clinical allergy and end-induction antiasparaginase (anti-ASNase) antibodies in children with high-risk acute lymphoblastic leukemia treated with pegylated (PEG) Escherichia coli ASNase and to determine whether they carry any prognostic significance. METHODS: Of 2057 eligible patients, 1155 were allocated to augmented arms in which PEG ASNase replaced native ASNase postinduction. Erwinia chrysanthemi (Erwinia) ASNase could be used to replace native ASNase after allergy, if available. Allergy and survival data were complete for 990 patients. End-induction antibody titers were available for 600 patients. RESULTS: During the consolidation phase, 289 of 990 patients (29.2%) had an allergic reaction. There were fewer allergic reactions to Erwinia ASNase than to native ASNase (odds ratio, 4.33; P < .0001) or PEG ASNase (odds ratio, 3.08; P < .0001) only during phase 1 of interim maintenance. There was no significant difference in 5-year event-free survival (EFS) between patients who received PEG ASNase throughout the entire study postinduction versus those who developed an allergic reaction to PEG ASNase during consolidation phase and subsequently received Erwinia ASNase (80.8% ± 2.8% and 81.6% ± 3.8%, respectively; P = .66). Patients who had positive antibody titers postinduction were more likely to have an allergic reaction to PEG ASNase (odds ratio, 2.4; P < .001). The 5-year EFS rate between patients who had negative versus positive antibody titers (80% ± 2.6% and 77.7% ± 4.3%, respectively; P = .68) and between patients who did not receive any ASNase postconsolidation and those who received PEG ASNase throughout the study (P = .22) were significantly different. CONCLUSIONS: The current results demonstrate differences in the incidence rates of toxicity between ASNase preparations but not in EFS. The presence of anti-ASNase antibodies did not affect EFS.

Inhibiting glutamine uptake represents an attractive new strategy for treating acute myeloid leukemia.

Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and protein synthesis inhibition downstream of mammalian target of rapamycin complex 1 (mTORC1) has shown promise as a possible therapy for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor formation in a mouse AML xenotransplantation model. l-asparaginase (l-ase) is an anticancer agent also harboring glutaminase activity. We show that l-ases from both Escherichia coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition correlates with their glutaminase activity levels and produces a strong apoptotic response in primary AML cells. We further show that l-ases upregulate glutamine synthase (GS) expression in leukemic cells and that a GS knockdown enhances l-ase-induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon l-ase treatment. These results suggest that l-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML.

Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry.

PURPOSE: Erwinia chrysanthemi L-asparaginase (ErA) is an enzyme commonly used in the treatment regimen for Acute Lymphoblastic Leukaemia (ALL). Biopharmaceutical products such as ErA must be monitored for modifications such as deamidation, typically using ion-exchange chromatography (IEX). Analysis of clinical-grade ErA using native IEX resolves a number of enzymatically-active, acidic variants that were poorly characterised. METHODS: ErA IEX variants were isolated and fully characterised using capillary electrophoresis (cIEF), LC-MS and LC-MS/MS of proteolytic digests, and structural techniques including circular dichroism, small-angle X-ray scattering (SAXS) and ion-mobility mass spectrometry (IM-MS). RESULTS: LC-MS, MS/MS and cIEF demonstrated that all ErA isolates consist mainly of enzyme lacking primary-sequence modifications (such as deamidation). Both SAXS and IM-MS revealed a different conformational state in the most prominent acidic IEX peak. However, SAXS data also suggested conformational differences between the main peak and major acidic variant were minor, based on comparisons with crystal structures. CONCLUSIONS: IEX data for biopharmaceuticals such as ErA should be thoroughly characterised, as the most common modifications, such as deamidation, may be absent.

Capillary isoelectric focusing of a difficult-to-denature tetrameric enzyme using alkylurea-urea mixtures.

Capillary isoelectric focusing (cIEF) is normally run under denaturing conditions using urea to expose any buried protein residues that may contribute to the overall charge. However, urea does not completely denature some proteins, such as the tetrameric enzyme Erwinia chrysanthemil-asparaginase (ErA), in which case electrophoresis-compatible alternative denaturants are required. Here, we show that alkylureas such as N-ethylurea provide increased denaturation during cIEF. The cIEF analysis of ErA in 8 M urea alone resulted in a cluster of ill-resolved peaks with isoelectric points (pI values) in the range 7.4 to 8.5. A combination of 2.0 to 2.2 M N-ethylurea and 8M urea provided sufficient denaturation of ErA, resulting in a main peak with a pI of 7.35 and an acidic species minor peak at 7.0, both comparing well with predicted pI values based on the sum of protein residue pKa values. Recombinant deamidated ErA mutants were also demonstrated to migrate to pI values consistent with predictions (pI 7.0 for one deamidation). The quantitation of ErA acidic species in samples from full-scale manufacturing (1.0-3.5% of total peak area) was found to be reproducible and linear. Use of alkylureas as denaturing agents in capillary electrophoresis and cIEF should be considered during biopharmaceutical assay development.

Safety profile of asparaginase Erwinia chrysanthemi in a large compassionate-use trial.

BACKGROUND: L-Asparaginase is an integral component of standard chemotherapy regimens for the treatment of acute lymphoblastic leukemia (ALL). Clinical hypersensitivity, a common reason for treatment discontinuation, has been reported in 10-30% of patients receiving Escherichia coli-derived asparaginase. After hypersensitivity, E. coli-derived asparaginase should be discontinued and an alternative asparaginase preparation, such as asparaginase Erwinia chrysanthemi, may be initiated. We conducted a compassionate-use study to collect additional safety information on asparaginase Erwinia chrysanthemi and to support FDA approval of the product. PROCEDURE: Patients with ALL or lymphoblastic lymphoma (LBL; N = 1368) who developed a hypersensitivity reaction (grade ≥2) to an E. coli-derived asparaginase participated in this trial. The recommended asparaginase Erwinia chrysanthemi dose was 25,000 IU/m(2) three days per week (Monday/Wednesday/Friday) for two consecutive weeks for each missed pegylated E. coli-derived asparaginase dose and 25,000 IU/m(2) for each missed nonpegylated asparaginase dose for the completion of their planned asparaginase treatment. RESULTS: Adverse event reports and/or case report forms were completed for 940 patients. The most common adverse event (AE) was hypersensitivity (13.6%). Eighteen patients (1.9%) died during the study. Most patients (77.6%) completed their planned asparaginase treatment with asparaginase Erwinia chrysanthemi. There was no apparent difference in the incidence of the most commonly reported AEs with asparaginase treatment by age, administration, or disease state. CONCLUSIONS: This study further established the safety profile of asparaginase Erwinia chrysanthemi in patients with ALL or LBL who had a hypersensitivity reaction to an E. coli-derived asparaginase.