Asparaginase Activity Research Articles

Recombinant Erwinia asparaginase (JZP458) in ALL/LBL: complete follow-up of the Children’s Oncology Group AALL1931 study

AbstractThe Children’s Oncology Group study AALL1931 investigated the efficacy and safety of recombinant Erwinia asparaginase (JZP458) in patients with acute lymphoblastic leukemia/lymphoblastic lymphoma and hypersensitivity reactions/silent inactivation to Escherichia coli–derived asparaginases. Each pegylated E coli asparaginase dose remaining in a patient’s treatment plan was replaced by intramuscular (IM) or IV JZP458 (6 doses) administered Monday/Wednesday/Friday (MWF). Three IM cohorts (1a [25 mg/m2 MWF], n = 33; 1b [37.5 mg/m2 MWF], n = 83; 1c [25/25/50 mg/m2 MWF], n = 51) and 1 IV cohort (25/25/50 mg/m2 MWF, n = 62) were evaluated. The proportion of patients maintaining nadir serum asparaginase activity (NSAA) levels of ≥0.1 IU/mL at the last 72 (primary end point) and 48 hours during course 1 was 90% (95% confidence interval [CI], 81-98) and 96% (95% CI, 90-100) in IM cohort 1c, respectively, and 40% (95% CI, 26-54) and 90% (95% CI, 82-98) in the IV cohort. Population pharmacokinetic modeling results were comparable with observed data, predicting the vast majority of patients would maintain therapeutic NSAA levels when JZP458 is administered IM or IV 25 mg/m2 every 48 hours, or IM 25/25/50 mg/m2 MWF, or with mixed administration of IM and IV (IV/IV/IM 25/25/50 mg/m2 MWF). Drug discontinuation occurred in 23% and 56% of patients in the IM and IV cohorts, respectively; 13% and 33% because of treatment-related adverse events (mainly allergic reactions and pancreatitis). JZP458 achieves therapeutic NSAA levels via multiple IM and IV dosing schedules based on a combination of observed and modeled data with a safety profile consistent with other asparaginases. This trial was registered at www.ClinicalTrials.gov as #NCT04145531.

EVALUATION OF THE EFFICACY OF ASPARAGINASE ADDED TO CHEMOTHERAPEUTIC DRUG REGIMEN IN ACUTE LYMPHOBLASTIC LEUKEMIA BY MINIMAL RESIDUAL DISEASE MEASUREMENT

B-ALL is the most common subtype of childhood Acute Lymphoblastic Leukemia (ALL), affecting approximately 85% of children worldwide. Minimal Residual Disease (MRD) monitoring during treatment of ALL is important for the prognosis of the disease. MRD monitoring, which enables early detection of relapse, determination of risk percentage and understanding the effectiveness of treatment, can be performed with multiple methods such as flow cytometry, qRT-PCR and NGS. Asparaginase is an enzyme that has been used in the treatment of ALL since the 1960s, converting asparagine to ammonia and aspartic acid, lowering serum asparagine levels and causing the death of malignant cells. In this study, we investigated the effect of asparaginase added to the ALL IC BFM 2009 treatment protocol in 62 B-ALL patients aged 0-18 years with MRD monitoring by flow cytometry. In our study, Escherichia Coli (E.Coli.) asparaginase was used primarily and PEG-asparaginase was used after allergy development. The effect of asparaginase on treatment was evaluated by evaluating asparagine concentration and asparaginase activity measured at TP1 (4th week after induction treatment) together with MRD levels. As a result of the study, it was observed that the added asparaginase positively affected the treatment and increased the negativity in MRD levels.

Evaluation of long-term stability of L-asparaginase encapsulated PLL-g-PEG nanoparticles in solution to aid in therapeutic delivery

Abstract L-asparaginase (L-ASNase) is a therapeutic enzyme that is widely used for the treatment of hematopoietic diseases such as acute lymphoblastic leukemia and lymphomas. L-ASNase destroys asparagine dependent tumors by degrading circulating L-asparagine and thereby destroying malignant cells. As a protein drug, L-ASNase carries a few inherent drawbacks including short circulating half-life, low stability, and low catalytic activity under physiological conditions. Moreover, due to the bacterial origin of L-ASNase used in treatments, there have been reports with high frequency of hypersensitivity reactions in patients. The use of this drug in adult cancer populations has largely been hindered not only due to its immunological side effects but also due to non-immunogenic toxicities such as pancreatitis, liver toxicities, coagulopathy, and neurotoxicity. Therefore, it is vital to find new methods to decrease its immunogenic/toxicity profile while increasing the stability and half-life. The purpose of this study is to achieve a new L-ASNase polymer nanocarrier to improve stability of the enzyme while masking it from the immune system of the host. We designed and characterized a nanoparticle (NP) where a poly-L-lysine-grafted-poly(ethylene) glycol co-polymer was used to encapsulate L-ASNase. The primary focus of the study was to evaluate the stability and encapsulation efficiency of this NP construct over time. There was no aggregation of NPs observed during the study period of 6 months in solution and NPs had a 0.436 mV surface charge. L-ASNase NPs showed a percent asparaginase activity of 31% compared to free L-ASNase. Under physiological conditions NPs were found to be intact and retained the encapsulated proteins for up to 6 months in solution. Together, these results demonstrate that L-ASNase loaded PLL-g-PEG NPs may serve as a fundamental platform to design nanocarriers to prolong stability in solution.

Screening and molecular identification of l-asparaginase producing bacteria with ansB gene from sewage water

Enzymes are vital biocatalysts facilitating biochemical reactions in organisms, playing pivotal roles in both biological systems and numerous industrial applications. This study focuses on the isolation and screening of asparaginase-producing bacteria from sewage water samples in Bhopal City. A modified M9 media, supplemented with asparagine and phenol red, was used to isolate asparaginase-producing bacteria. Out of the ten samples, seven exhibited asparaginase activity. Two bacterial strains, identified as Bacillus tropicus (S-1) and Priestiaveravalensis (S-2), were found to harbor the ansB gene. Sequence analysis revealed a 96.52% and 95.09% resemblance to Bacillus tropicus MCCC 1A01406 and Priestiaveravalensis strain SGD-V-76, respectively. These findings highlight the potential of these bacterial strains in producing L-asparaginase, emphasizing their significance in pharmaceutical applications, particularly in cancer treatment.

Effects of Different Organic Fertilizer Substitutions for Chemical Nitrogen Fertilizer on Soil Fertility and Nitrogen Use Efficiency of Foxtail Millet

Conventional fertilizer management can destroy the structure of soil. Replacing chemical fertilizers with organic fertilizers can improve soil quality and nitrogen use efficiency. We aimed to study the effects of organic fertilizer substitutions for chemical nitrogen fertilizer on soil fertility and nitrogen use efficiency in order to clarify the effectiveness of the available nutrient management measures in improving soil quality and increasing foxtail millet yield. A field experiment was carried out over two consecutive years, and a total of six treatments were set up: no fertilizer (CK), chemical nitrogen fertilizer alone (N), the substitution of 25% of chemical nitrogen fertilizer with bio-organic fertilizer (N25A1), the substitution of 25% of chemical nitrogen fertilizer with fermented mealworm manure (N25B1), the substitution of 50% of chemical nitrogen fertilizer with bio-organic fertilizer (N50A2), and the substitution of 50% of chemical nitrogen fertilizer with fermented mealworm manure (N50B2). The results of this study show the following: (1) Compared with chemical nitrogen fertilizer, the substitution of organic fertilizer for nitrogen fertilizer reduced the bulk density and solid phase of the soil, and it increased the total porosity, water content, liquid phase, and gas phase of the soil. (2) Compared with nitrogen fertilizer, the use of an organic fertilizer increased the contents of nitrate nitrogen, ammonium nitrogen, and total nitrogen in the soil by 13.59~52.56%, 4.47~18.27%, and 4.40~12.09%, respectively. The content of alkaline nitrogen increased by 1.70~32.37%, and the contents of soil available potassium, available phosphorus, and organic matter also increased. (3) The activities of sucrase, urease, glutaminase, and asparaginase were improved by replacing chemical nitrogen fertilizer with organic fertilizer. The N25 treatments performed better than the N50 treatments, and fermented mealworm manure performed better than biological organic fertilizer. (4) A moderate application of organic fertilizer (N25) can increase the grain yield, ear weight, grain weight, and 1000-grain weight of foxtail millet, whereas excessive application of organic fertilizer (N50) can reduce foxtail millet yield. (5) Replacing chemical nitrogen fertilizer with organic fertilizer can improve the agronomic use efficiency, physiological efficiency, biased productivity, harvest index, and apparent use efficiency of nitrogen fertilizer. In this study, the substitution of 25% of chemical nitrogen fertilizer with fermented mealworm manure was the best combination for restoring crop productivity and soil quality.

Long-Term Cropping Management Practices Affect the Biochemical Properties of an Alabama Ultisol

Interest in improving the long-term sustainability of agricultural production systems has focused on identifying management practices that promote soil health. No tillage, cover cropping, and amending soils with broiler (Gallus gallus domesticus L.) litter are commonly adopted conservation practices that have been shown to improve soil fertility and crop yield. However, the overall influence of these conservation practices on soil health in the southeastern US are not well understood. Thus, a study was conducted to evaluate the influence of tillage, broiler litter (BL) applications, and cropping systems on soil biochemical properties. Soils were collected from field research plots under long-term management (>than 25 years of tillage, 15 years of broiler litter application, and 15 years of cropping system). Soil microbial biomass, C, N, and P, amidohydrolases, and dissolved organic matter (DOM) were evaluated as indicators of soil health. Adopting tillage and BL into the agricultural management system modified the biochemical parameters of the soils evaluated. Most of these modifications occurred in the 0–5 cm depth. Higher microbial biomass carbon (MBC; 85%) and nitrogen (MBN; 10%) and enzyme activities of asparaginase (65%) and glutaminase (70%) were observed in the 0–5 cm depth under no tillage (NT) compared to conventional tillage (CT), indicating greater biological activities were established in these soil ecosystems. Broiler litter applications increased microbial biomass N and activities of asparaginase and glutaminase in both soil depths. In addition, microbial biomass phosphorus (MBP) was increased following BL application in the 0–5 cm depth. The results suggest that long-term management of NT and BL additions can improve the health of eroded southeastern US soils by altering the soil biochemical parameters.

Pharmacokinetics of PEGasparaginase in Infants with Acute Lymphoblastic Leukemia.

PEGasparaginase is known to be a critical drug for treating pediatric acute lymphoblastic leukemia (ALL), however, there is insufficient evidence to determine the optimal dose for infants who are less than one year of age at diagnosis. This international study was conducted to identify the pharmacokinetics of PEGasparaginase in infants with newly diagnosed ALL and gather insight into the clearance and dosing of this population. Infants with ALL who received treatment with PEGasparaginase were included in our population pharmacokinetic assessment employing non-linear mixed effects modelling (NONMEM). 68 infants with ALL, with a total of 388 asparaginase activity samples, were included. PEGasparaginase doses ranging from 400 to 3,663IU/m2 were administered either intravenously or intramuscularly. A one-compartment model with time-dependent clearance, modeled using a transit model, provided the best fit to the data. Body weight was significantly correlated with clearance and volume of distribution. The final model estimated a half-life of 11.7days just after administration, which decreased to 1.8days 14days after administration. Clearance was 19.5% lower during the post-induction treatment phase compared to induction. The pharmacokinetics of PEGasparaginase in infants diagnosed under one year of age with ALL is comparable to that of older children (1-18years). We recommend a PEGasparaginase dosing at 1,500IU/m2 for infants without dose adaptations according to age, and implementing therapeutic drug monitoring as standard practice.

Desensitization using pegaspargase in the era of commercially available Erwinia: A single-institution report on efficacy, cost, and resource utilization.

Pegaspargase is a therapeutic enzyme that is utilized in treatment regimens targeting pediatric acute lymphoblastic leukemia. However, many patients experience hypersensitivity reactions, requiring discontinuation of the therapy. Historically, this necessitated switching to an alternative form of the drug, most commonly asparaginase Erwinia chrysanthemi; however, in recent years this was difficult due to drug shortages and eventually commercial discontinuation. We report here our experience performing pegaspargase desensitizations in patients with prior hypersensitivity reactions. Patients with a clinical hypersensitivity reaction to pegaspargase were identified. When due for their next dose, patients were admitted to the pediatric intensive care unit, bone marrow transplant unit, or oncology unit, and underwent desensitization utilizing a rigorous premedication and multistep dilution-based protocol. Serum asparaginase activity levels were drawn after desensitization to assess for therapeutic levels of enzyme activity. We identified 11 patients who underwent a total of 33 desensitizations to pegaspargase and calaspargase pegol-mknl. No patients experienced clinically significant hypersensitivity reactions necessitating stopping the infusion, nor administration of rescue medications. All serum asparaginase activity levels collected demonstrated enzyme activity levels above predefined therapeutic thresholds. Cost analysis revealed substantial savings when patients received asparaginase desensitization over the now commercially available asparaginase E. chrysanthemi (recombinant) rywn. Performing desensitization to pegaspargase in the pediatric acute lymphoblastic leukemia population is feasible, safe, and effective. It is financially advantageous over available alternative approaches, and requires fewer injections and presentations to care.

Continuous PEGasparaginase Dosing Reduces Hypersensitivity Reactions in Pediatric ALL: A Dutch Childhood Oncology Group ALL11 Randomized Trial.

The primary objective of this randomized study was to determine whether a continuous dosing schedule (without the asparaginase-free interval) would result in less hypersensitivity reactions to PEGasparaginase (PEGasp) compared with the standard noncontinuous dosing schedule. Eight hundred eighteen patients (age 1-18 years) with ALL were enrolled in the Dutch Childhood Oncology Group-ALL11 protocol and received PEGasp. Three hundred twelve patients stratified in the medium-risk arm were randomly assigned to receive 14 individualized PEGasp doses once every two weeks in either a noncontinuous or continuous schedule after the first three doses in induction (EudraCT: 2012-000067-25). Hypersensitivity reactions were defined as allergies, allergic-like reactions, and silent inactivation. Secondary end points were other asparaginase-related toxicities, asparaginase activity and antibody levels, and outcome. During induction, 27 of 818 patients (3.3%) experienced hypersensitivity reactions. After random assignment, 4 of 155 (2.6%) in the continuous treatment arm versus 17 of 157 (10.8%) patients in the noncontinuous treatment arm had hypersensitivity reactions (P < .01), of which two (1.3%) versus 13 (8.3%) were inactivating reactions (P < .01). The occurrence of inactivating hypersensitivity reactions was seven times lower in the continuous arm (odds ratio, 0.15 [0.032-0.653]). In addition, antibody levels were significantly lower in the continuous arm (P < .01). With exception of a lower incidence of increased amylase in the continuous arm, there were no significant differences in total number of asparaginase-associated toxicities between arms. However, the timing of the toxicities was associated with the timing of the asparaginase administrations. No difference in 5-year cumulative incidence of relapse, death, or disease-free survival was found between both treatment arms. A continuous dosing schedule of PEGasp is an effective approach to prevent antibody formation and inactivating hypersensitivity reactions. The continuous PEGasp schedule did not increase toxicity and did not affect the efficacy of the therapy.

Implementation of plate reader-based indooxine and Nessler protocols for monitoring L-asparaginase serum activity in childhood acute lymphoblastic leukaemia.

Monitoring the blood serum activity of L-asparaginase in children with acute lymphoblastic leukaemia (ALL) has been highly recommended to detect enzyme inactivation that can cause relapse and to avoid unwanted toxicity. Nevertheless, perhaps at least partially due to the lack of clinically approved commercially available kits or standardized and independently reproduced and validated in-house protocols, laboratory assay-based determination of the optimal doses of L-asparaginase is not carried out routinely. In this study, we adapted previously published protocols for two plate reader-based colorimetric methods, indooxine and Nessler, to measure asparaginase activity. Mock samples with dilutions of the enzyme for initial optimization steps, and patient samples were used as a proof of principle and to compare the two protocols. For the first time the indooxine and the Nessler methods are adapted for a plate reader and L-asparaginase serum activity levels are compared by both protocols. Passing-Bablok and Bland-Altman's statistical analyses found very little difference, strong correlation (r = 0.852), and bias of only 6% between the data from the two methods when used for fresh patient samples. Furthermore, we demonstrate that the Nessler method could also be applied for frozen sera as the results, compared to fresh samples, showed little difference, strong correlation (r = 0.817), and small bias (9%). We successfully adapted and validated two methods for measuring L-asparaginase activity in cALL and provided the most detailed description to date on how to reproduce and implement them in other clinical laboratories.

Asparaginase-Associated Toxicities and Hypersensitivity Reactions in Pediatric and Adolescent Down Syndrome Patients with Acute Lymphoblastic Leukemia or Lymphoma

Children with Down syndrome acute lymphoblastic leukemia (DS-ALL) have worse prognosis compared to those with non-DS-ALL, largely due to higher treatment related mortality. Pegaspargase (PEG) is integral in protocols for the treatment of pediatric ALL and lymphoblastic lymphoma (LBL), and full exposure to all doses of PEG can be limited by toxicities, including hypersensitivity reactions (HSR) due to the production of anti-asparaginase antibodies rendering PEG therapy ineffective. To date, minimal data has been reported on the use of PEG in children with DS-ALL/LBL. We conducted a prospective, multicenter study to evaluate the efficacy of premedicating pediatric and adolescent ALL/LBL patients receiving PEG with antihistamines-H1 and -H2 blockers with subsequent therapeutic drug monitoring (TDM) to better identify patients with true HSR versus those with non-antibody mediated infusion reactions. Here, we report on the incidence of HSRs and PEG-associated toxicities in the cohort of children with DS-ALL/LBL. Eligible patients from six centers received PEG, as per a Children's Oncology Group protocol, beginning in induction therapy. An H1 blocker (diphenhydramine) and H2 blocker (ranitidine, famotidine or cimetidine) were administered prior to PEG administration in consolidation and subsequent cycles. TDM was performed at 7 and 14 days post each PEG. PEG was administered at 2500 International units/m 2/dose that was capped for obesity or age &amp;gt; 22 years. Patients were monitored for HSR and other PEG-related toxicities using CTCAE version 5 criteria. Therapeutic asparaginase activity (AA) was defined as ≥ 0.1 IU/mL (≥100 IU/L) at 7 days post infusion and greater than the lower level of quantification at 14 days. We observed for silent inactivation (subtherapeutic levels without symptoms) and accelerated clearance (levels below quantification at 14 days without symptoms). Descriptive statistics summarized patient characteristics and mixed effects modeling procedure assessed mean AA levels at 7 and 14 days with specification of repeat measures and compound symmetry covariance structure. Out of 148 patients, 9 DS (8 B-ALL, 1 B-LBL) were enrolled between 10/2019 - 3/2022. The median age at diagnosis was 5.0 years [IQR: 4.3, 14.9], 4 were National Cancer Institute (NCI) High Risk (HR) at diagnosis, 4 were Hispanic and none were obese. ALL 9 DS patients received a total of 26 doses of PEG at the standard dose without dose capping required. The mean unadjusted AA levels at 7 and 14 days post PEG were 0.76 IU/ml (SE=0.11) and 0.54 IU/ml (SE=0.11), respectively, p&amp;lt;.001. None of the DS patients experienced HSR or silent inactivation. Accelerated clearance was observed in 1 patient. Nineteen episodes of grade &amp;gt;3 PEG-associated toxicity were observed in 5 DS-ALL patients (5/9; 55.6%). PEG-associated toxicity was observed more frequently in older patients whose mean age was 13 years (SD=6.2) versus 4.3 years in those without toxicity (SDE=0.9), p=.028. Four of the DS-ALL who experienced toxicities were NCI HR at diagnosis and &amp;gt; 10 years of age. For the 19 episodes of toxicities: 6 occurred during induction, 9 during consolidation, and 4 during delayed intensification. The most common toxicity observed was hepatoxicity (increased alanine transaminase) which was experienced by 3 DS-ALL patients. There were no grade &amp;gt; 3 hyperbilirubinemia. All 4 DS-ALL patients who experienced PEG toxicity during induction also had toxicity with subsequent cycles. One DS-ALL patient had a dose limiting toxicity (grade 3 pancreatic pseudocyst), requiring discontinuation of subsequent PEG therapy. There were no grade 4 or 5 toxicities observed. All 4 DS patients who did not experience toxicities were NCI Standard Risk patients. All DS patients had undetectable minimal residual disease (MRD) that was &amp;lt; 0.01 at the end of induction therapy. We noted that in this small DS-ALL/LBL cohort, none of the patients experienced HSR, and all achieved therapeutic AA at both time points of 7 and 10 days post PEG. Five (55.6%) of the DS-ALL patients experienced at least one grade &amp;gt; 3 asparaginase toxicity, and they were older than those without toxicity. Most toxicity occurred during the consolidation phase and no grade 4 or 5 toxicities were observed. Given the PEG-associated toxicity observed in DS ALL/LBL, we are conducting pharmacokinetic analysis to propose a lower dose of PEG which may minimize toxicity while maintaining therapeutic efficacy.

Efficacy and Safety of Recombinant Erwinia Asparaginase (JZP458) in Acute Lymphoblastic Leukemia (ALL) or Lymphoblastic Lymphoma (LBL): Complete Follow-up of the Children's Oncology Group (COG) AALL1931 Study

Background: L-asparaginase is an important component of multi-agent treatment regimens for pediatric and adult patients with ALL/LBL. However, hypersensitivity reactions to E. coli-derived asparaginases often lead to treatment delay or discontinuation. JZP458, a recombinant Erwinia asparaginase (ASP) derived from a Pseudomonas fluorescens expression platform, was evaluated in study AALL1931, a 2-part, open-label, phase 2/3 trial conducted with the Children's Oncology Group investigating efficacy, safety, and pharmacokinetics (PK) of JZP458 in patients with ALL/LBL (ClinicalTrials.gov ID: NCT04145531). Based on interim results from part A of AALL1931, intramuscular (IM) JZP458 (Rylaze®) was approved by the US Food and Drug Administration for treatment of ALL/LBL in June 2021. Here, we report the efficacy and safety of JZP458 at the completion of AALL1931. Methods: Eligible patients with ALL/LBL who daeveloped hypersensitivity (grade ≥3 allergic reaction or silent inactivation) to E. coli-derived pegaspargase received JZP458 as part of their multi-agent treatment plan. Each dose of pegaspargase was substituted with 6 doses of JZP458 administered either IM (part A) or intravenously (IV, part B) on Monday/Wednesday/Friday (MWF) over 2 weeks, defined as 1 course. The study enrolled 3 IM cohorts [1a (25 mg/m 2 MWF), 1b (37.5 mg/m 2 MWF), and 1c (25/25/50 mg/m 2 MWF)], and 1 IV cohort (25/25/50 mg/m 2 MWF). Efficacy was assessed by proportion of patients maintaining therapeutic nadir serum ASP activity (NSAA) ≥0.1 IU/mL at the last 72-hour (primary endpoint) or 48-hour (key secondary endpoint) timepoint during course 1. A population PK (PPK) model was developed based on serum ASP activity (SAA) data from AALL1931 to characterize the PK of JZP458 and simulations using the PPK model were performed to provide additional information on alternative dosing regimens. Results: At the final cutoff date of November 22, 2022, a total of 167 patients received IM JZP458 (1a, n=33; 1b, n=83; 1c, n=51) and 61 patients received IV JZP458. The median (range) of JZP458 courses received was 5 (0, 14), 4 (0, 15), 5 (1, 10), and 3 (0, 15) for IM cohorts 1a, 1b, 1c, and IV cohort, respectively. Table 1 shows the proportion of patients who achieved therapeutic NSAA levels and the mean SAA levels per cohort in course 1. With IM cohort 1c (25/25/50 mg/m 2 MWF), the proportion (95% CI) of patients achieving NSAA levels ≥0.1 IU/mL at 72 and 48 hours were 90% (81, 98) and 96% (90, 100), respectively. Treatment-related adverse events (TRAEs) ≥ grade 3 occurred in 126/228 (55%) patients; but no TRAE led to death. Table 2 shows the most commonly reported (≥10% of patients) nonhematologic TRAEs. A total of 22 patients (13%) across all IM cohorts (2 patients in 1a, 14 in 1b, and 6 in 1c) and 20 patients (33%) in the IV cohort experienced a TRAE leading to treatment discontinuation.The most common TRAEs leading to treatment discontinuation were pancreatitis (6%) in the IM cohort, and drug hypersensitivity (15%) in the IV cohort. PPK model-based simulations predicted that therapeutic NSAA levels are achieved in the vast majority of patients when JZP458 is administered IM at 25 mg/m 2 every 48 hours (7 doses) or 25/25/50 mg/m 2 MWF (6 doses), when JZP458 is administered IV at 25 mg/m 2 every 48 hours (7 doses), or when JZP458 is administered IV at 25 mg/m 2 M/W and IM at 50 mg/m 2 F (6 doses). Conclusions: Results at the completion of AALL1931 in patients with ALL/LBL and allergic reactions or silent inactivation to E. coli-derived asparaginase show the safety profile of JZP458 is consistent with other asparaginases, with no new adverse safety signals. Treatment-related discontinuation rates with IV JZP458 were greater than with IM, primarily due to increased incidence of hypersensitivity/infusion-related reactions along with nausea and vomiting; however, the study was not statistically powered to compare different dosing cohorts. Observed and PPK modeling data demonstrate JZP458 achieves therapeutic NSAA levels via multiple IM and IV dosing schedules, with IM dosing having more sustained SAA, and therefore providing flexibility to patients and physicians.

Dissecting dual specificity: Identifying key residues in L-asparaginase for enhanced acute lymphoid leukemia therapy and reduced adverse effects

L-asparaginase from Escherichia coli (EcA) has been used for the treatment of acute lymphoid leukemia (ALL) since the 1970s. Nevertheless, the enzyme has a second specificity that results in glutaminase breakdown, resulting in depletion from the patient's body, causing severe adverse effects. Despite the huge interest in the use of this enzyme, the exact process of glutamine depletion is still unknown and there is no consensus regarding L-asparagine hydrolysis. Here, we investigate the role of T12, Y25, and T89 in asparaginase and glutaminase activities. We obtained individual clones containing mutations in the T12, Y25 or T89 residues. After the recombinant production of wild-type and mutated EcA, The purified samples were subjected to structural analysis using Nano Differential Scanning Fluorimetry, which revealed that all samples contained thermostable molecules in their active structural conformation, the homotetramer conformation. The quaternary conformation was confirmed by DLS and SEC. The activity enzymatic assay combined with molecular dynamics simulation identified the contribution of T12, Y25, and T89 residues in EcA glutaminase and asparaginase activities. Our results mapped the enzymatic behavior paving the way for the designing of improved EcA enzymes, which is important in the treatment of ALL.

To Reverse Sensitization By Co-Administration of Bortezomib and Rituximab with Conventional Chemotherapy for Patients Who Developed Neutralizing Hypersensitivity Reactions Against Asparaginase

Background: Acute lymphoblastic leukemia (ALL) patients undergoing Pegylated asparaginase (Peg-asp) or Erwinia-asparaginase (Erw-asp) preparation therapy may experience silent inactivation and allergy to asparaginase (ASP), leading to decreased therapeutic efficacy. How to treat ASP sensitized patients, especially those who are allergic to both Peg-asp and Erw-asp, without compromising the treatment outcome remains a problem for hematologists.This study aims to investigate whether the sensitization to Peg-asp can be reversed through the co-treatment of bortezomib and rituximab with conventional chemotherapy for patients hypersensitive to both Peg-asp and Erw-asp. Method: ALL Patients who developed silent inactivation and allergic reaction to both Peg-asp and Erw-asp enrolled in the study. The study featured 2 courses of combined conventional chemotherapy with rituximab and bortezomib co-treatment. In the second course of desensitization, Peg-asp was re-exposed according to the severity of previous allergic reaction against Peg-asp. For those with silent inactivation or previous allergic reactions of grade ≤ III, a split intramuscular injection was administered (the first dose was given at 1/10th of the required dosage, and then the remaining dose was given at an interval of 2 hours if there was no allergic reaction). Patients with grade IV previous allergic reactions underwent a standard 3-bag desensitization procedure. ASP activity was measured on days 1, 3, 7, 10, 14, and 21 after the first Peg-asp re-challenge. If re-exposure to the first dose did not result in an allergic reaction, a subsequent intramuscular single full dose was administered based on the results of ASP activity monitoring. Primary endpoint: to observe the percentage of allergic reaction on re-exposure and the duration of sustained ASP activity. Result: From August 2020 to May 2023, 10 patients of ALL who developed a neutralizing allergy or silent inactivation to both Peg-asp and Erw-asp preparation were recruited to the study. Among them, six cases were B-ALL, and four were T-ALL. The median age of the participants was 8 years, with seven boys and three girls. Seven children experienced Peg-asp neutralizing allergy or silent inactivation during remission induction, while three children experienced it during the interphase treatment. Eight of them had clinical manifestations of allergy and two had silent inactivation. During the second desensitization course, none of the children showed any allergic reaction after the first re-exposure to Peg-asp. Three children maintained &amp;gt;3 weeks of therapeutic activity after the first Peg-asp re-exposure, while one child maintained 14 days, and three children maintained 10 days. Three children detected Asp activity below 100U/L on day 3 monitoring. After two courses of desensitization, the patients returned to the original chemotherapy regimen. Nine patients (90%) maintained activity above 0.1IU/ML for more than three weeks after subsequent Peg-asp administration. One child developed pancreatitis after the last dose of Peg-asp but recovered soon. Another child experienced silent inactivation and was discontinued from all ASP administration. During desensitization therapy, eight children developed febrile neutropenia. Three were recovered with outpatient antibiotic treatment, while five required hospitalization for 5-7 days. As of July 30, 2023, all ten children survived without any adverse events, with a median follow-up time of 28 months from the start of desensitization therapy. Conclusion： Rituximab and bortezomib co-treatment effectively reverse ASP sensitization and restore the sustained potent activity of Peg-asp. This co-treatment is a safe and promising desensitization therapy after ASP sensitization in ALL patients.

Overcoming Venetoclax (Ven) Resistance through Glutamine (Gln) Depletion: Final Analysis of the Phase 1 Trial of Ven and Pegcrisantaspase (PegC) Combination in Relapsed and Refractory (R/R) Acute Myeloid Leukemia (AML)

Background: Ven-based therapies have been an advancement in the front-line treatment of AML; however development of resistance is common and the efficacy of Ven-containing regimens in R/R AML has been far less promising. We reported in vitro and in vivo depletion of Gln induced by long-acting Erwinia asparaginase, PegC, inhibited proliferation of complex karyotype (CK) AML and synergistically enhanced the antiapoptotic activity of Ven-mediated antagonism of Bcl-2 by decreasing the expression of proteins such as Mcl-1, whose translation is cap-dependent. Here we report the final results of our phase 1 clinical trial (NCT04666649) of VenPegC for treatment of R/R AML including patients (pts) previously treated with Ven. Methods: Pts received Ven 400 mg on D1-28 and PegC (500, 750, 1000 IU/m 2 in cohorts 1-3, IV) D1 &amp; D15 every 4 weeks (wk). Primary study endpoints were incidences of regimen limiting toxicities (RLT) and RP2D. Secondary endpoints included rate of composite complete remissions. Correlative studies included monitoring plasma amino acid levels and asparaginase activity, and expression of proteins regulating cap-dependent mRNA translation in bone marrow (BM) mononuclear cells pre- and post-VenPegC treatment. Results: As of July 31, 2023, 26 pts were enrolled of whom 24 received at least 1 dose of PegC and 17 completed at least 1 cycle of PegC (2 doses). The median age was 61 (range 24-79), 16 (62%) were female; 9 (35%) had CK-AML and 5 (19%) had TP53 mutations; 18 (69%) were previously treated with Ven. Median prior lines of treatment were 2 (range 1-5). No RLT was observed in cohort 1 (n=3). Cohort 2 was expanded to 6 pts due to transient asymptomatic hypertriglyceridemia. Two pts in cohort 3 (n=3) developed grade 3 hyperbilirubinemia determined as RLT. Expansion cohort included 11 pts who were treated at MTD/RP2D (PegC 750 IU/m 2 plus Ven 400 mg) given as out-pt without a need for hospitalization. No pancreatitis, thrombohemorrhagic events, or silent inactivation were observed in the entire study population. Overall response rate (ORR) for pts who received ≥1 cycle of PegC was 47% (8/17), Fig 1. CR+ CRh+CRi rate was 29% (5/17); 1 pt had a partial remission with full count recovery and is currently receiving cycle 6; 2 pts had stable disease. Four pts showed hematologic improvement. Twelve (71%) pts who received ≥1 cycle of PegC were previously treated with Ven and were Ven-refractory. ORR in Ven-refractory pts was 42% (5/12); of the 5 pts who harbored a RAS or PTPN11 mutation and previously treated with Ven, 60% responded. Remarkably, all pts (3/3) who had a RUNX1 mutation achieved a CR; Pt 6 (relapsed AML with NRAS/cKIT/RUNX1 mutations) achieved an MRD-negative CRh and underwent an allogeneic stem cell transplant (alloHSCT); Pt 12 (primary refractory AML with TP53/NRAS/RUNX1 mutations) achieved a clearance of BM blasts after 1 dose of PegC and achieved MRD-negative CR at the end of cycle 1, and Pt 15 (post-alloHSCT relapsed AML with NRAS/PTPN11/CSF3R/RUNX1 mutations) achieved a CRi and received 10 cycles of VenPegC. Pt 31 (post-alloHSCT relapsed AML with FLT3-ITD) who had persistent disease after gilteritinib achieved an MRD-negative CRi, pending 2 nd alloHSCT. Nadir plasma asparaginase activity of &amp;gt;0.1 IU/mL was achieved in cycle 1 for all pts who received ≥1 cycle of VenPegC. All pts with available results achieved undetectable asparagine (Asn) level by day 7 and Asn remained undetectable throughout the study. Plasma Gln reduction from baseline occurred in all pts (except 1) within cycle 1. Pts in cohort 1 reached nadir Gln after initial PegC exposure in an average of 22 days compared to 16 days in cohort 2. In general, pts who achieved CR had the lowest absolute plasma Gln levels. Available Glu and Gln are shown in Fig.1 for pts who achieved a CR/PR. Out of the 8 assessed pts (4 responders &amp; 4 non-responders), VenPegC inhibited cap-dependent translation after 1 cycle in responders, as evidenced by decreased phosphorylated eIF4E (active, pheIF4E) in 4/4 pts and decreased phosphorylated 4EBP1 (inactive, ph4EBP1), a negative regulator of eIF4E, in 2/4 pts. Conversely, pheIF4E and ph4EBP1 expression was increased in 3/4 and 4/4 of the non-responders, respectively (Fig.2). Conclusion: VenPegC is a novel regimen that can induce complete remission in some heavily pretreated R/R AML patients, even with previous exposure to Ven. Pts with RUNX1 mutation, whose translation depends on pheIF4E, may benefit further from this regimen.

Development of a Novel L-Asparaginase with Enhanced Properties Using Ancestral Sequence Reconstruction

The overall survival of children with acute lymphoblastic leukemia (ALL) now exceeds 90% due to the precise disease risk stratification and intensification of multi-agent chemotherapy regimens. L-asparaginase (L-ASNase) is an indispensable biotherapeutic enzyme used in treatment of pediatric ALL. Discontinuation of L-ASNase treatment due to immunological or adverse reactions leads to significantly inferior disease-free survival (73% ±7% vs 92% ± 2%; P &amp;lt;0.01, Gupta et al. JCO 2020). Clinical L-ASNases are bacterial in origin, derived from either Escherichia coli or Erwinia chrysanthemi and are therefore highly immunogenic. Additionally, emerging data suggests that the concurrent glutaminase activity seen in bacterial asparaginases exacerbates its liver and pancreatic toxicity, especially in adults thereby limiting its extensive use beyond children. Therefore, finding alternative sources with preferable biochemical features remains paramount. The inferior catalytic properties of human L-ASNase restricts its functionality in ALL treatment. However, characterization has shown that guinea pig (GP) L-ASNase demonstrates significant anti-tumor properties and improved enzyme kinetics despite a 69.8% amino acid sequence identity with human L-ASNase, as compared to 30% for bacterial L-ASNases. Additionally, guinea pig L-ASNase has no glutaminase activity. Thus, to overcome the major deficiencies of bacterial L-ASNase and develop a more humanized form of L-ASNase we performed ancestral sequence reconstruction (ASR). ASR is a protein drug discovery / optimization platform that predicts ancient DNA and protein sequences from extant sequences. This approach permits higher-resolution mapping potentially highlighting functional resides. The objective therefore is to exploit functional diversity refined by natural selection using ASR to identify therapeutic variants with superior properties. Recent work on guinea pig (GP) L-ASNase (Schalk et al. J Biol Chem 2014) identified a low micromolar (μM) Michaelis constant (Km) at 57.7 ± 6.4 μM essential for hydrolyzing the extra-cellular pool of asparagine ~50 μM in human blood; thus, equipping us with the ideal mammalian L-ASNase ortholog to serve as a template to resurrect ancient ASNase sequences. Ancestral L-ASNase sequences were generated utilizing 54 extant L- asparaginase sequences, aligned using MUSCLE and an evolutionary tree inferred using MrBayes. A total of 53 ancestral nodes were identified, and the ten ancestral L-ASNase variants spanning the ancient primate and guinea pig (GP) lineage were resurrected. The sequence identity of these ancestral L-ASNase variants ranged from 81 to 98% when compared to human L-ASNase. E. coli codon optimized complementary DNA (cDNA) sequences were synthesized and subcloned intoan expression vector. Correctly cloned plasmids were transformed into E. coli BL21 (DE3) cells for protein expression. An-ASNase candidates were isolated by Ni 2+ affinity chromatography and then further purified by size exclusion chromatography. Asparaginase activity of the An-ASNase candidates were determined via a modified Nessler's reagent assay by using a continuous spectroscopic enzyme-coupled assay. Three of the ancestral L-ASNase candidates, An-88, An-104 and An-107 demonstrated excellent asparaginase activity when tested at an enzyme concentration of 0.1 mg/mL and an excess substrate concentration, with activity within a comparable range to the clinically relevant E. coli and Erwinia asparaginases. An-88 had 81% similarity whereas both An-104 and An-107 ASNases had 88% identity to human L-ASNase, which is in stark contrast to the only 30% similarity seen with bacterial L-ASNases. Detailed enzymatic characterization of these three lead drug candidates is currently being performed. Preliminary cytotoxicity data testing the ancestral L-ASNase candidates against a T-cell ALL cell line CCRF-CEM showed an expected dose response, with An-107 showing the highest cytotoxicity. Our findings demonstrate that ASR is an efficient protein drug design/optimization platform that we have now extended beyond our previous work with recombinant coagulation factors (Zakas et al. Nat Biotech 2017, Knight et al. Blood Adv 2021) to therapeutic enzymes; thereby, enabling the creation of novel recombinant L-ASNases with translational implications due to improved enzymatic specificity and reduced toxicity.

A Real-World, Single Institution, Post-Market Comparison of Pegaspargase and Calasparagase Pegol-Mknl Therapeutic Drug Monitoring and Toxicity

Introduction: Calaspargase pegol-mknl (CalPEG) was FDA-approved in December 2018 as a component of a multi-agent chemotherapeutic regimen for acute lymphoblastic leukemia/lymphoma (ALL/LLy) in pediatric patients and adolescent and young adults (AYA). It has recently been incorporated into national pediatric ALL consortia clinical trials. The pharmacokinetics and toxicity profiles were reported in two clinical trials (AALL07P4; Angiolillo et al, J Clin Oncol, 2014 and DFCI 11-001; Vrooman et al, J Clin Oncol, 2021), with similar safety profiles to historical pegaspargase (PEGasp). There is a paucity of published data regarding serum asparaginase activity (SAA) level variation or toxicity between CalPEG and PEGasp since FDA-approval. This is timely given the recent transition in asparaginase product availability, which may impact resource utilization, comprehensive drug safety monitoring, and clinical practice guidelines. Methods: We conducted a single institution retrospective review of children and AYA with ALL/LLy who received an asparaginase product during upfront or relapse therapy between January 2022 to June 2023 as part of our chemotherapy quality and safety monitoring (Table). Patients received either PEGasp, CalPEG, and/or asparaginase Erwinia chrysanthemi recombinant (Rylaze) and had correlative SAA documented by a validated assay (NEXT Molecular Analytics; Chester, VA). The Common Terminology Criteria for Adverse Events version 5.0 was used to retrospectively grade asparaginase-related toxicities, including pancreatitis (≥ grade 3), venous thromboembolism (VTE; ≥ grade 2), hyperbilirubinemia (total bilirubin ≥ 5 mg/dL), allergy (≥ grade 3), and silent inactivation (SI, SAA &amp;lt; 0.025 IU/mL within 7 days post-treatment without allergic symptoms). Fisher's exact and Wilcoxon rank-sum tests were used to compare patient characteristics and toxicity incidence with pairwise post-hoc comparisons. P values were two-sided and considered significant if &amp;lt; 0.05. Generalized linear modeling (GLM) was used to model SAA over time and interactions among asparaginase product, toxicity, and immunophenotype. Results: One hundred twenty-five patients received either PEGasp, CalPEG, and/or Rylaze. (Table). Frequency of total asparaginase-related toxicity was comparable between CalPEG (27.7%) and PEGasp (27.4%). However, the incidence of pancreatitis was significantly higher in patients who received CalPEG (12.8%) compared to PEGasp (2.4%), P=0.025, with an Odds Ratio (OR) 5.91. T-ALL/LLy patients who received CalPEG had an increased incidence of both pancreatitis and VTE (33.3% each), with statistically significant pairwise interactions to PEGasp and/or B-ALL cohorts. Conversely, allergy was significantly higher in PEGasp (17.9%) compared to CalPEG (4.3%), P=0.03, with an OR 4.46. SI was found in 5.8% of PEGasp with none yet identified in CalPEG patients. The median peak SAA level over the first 10 days was not different among patients with adverse events; nor were median SAA levels higher in CalPEG patients, despite increased incidence in treatment-related toxicity (Figure). GLM fit slopes were predictably less negative in CalPEG compared to those of PEGasp; patients with toxicity had a larger modeled area under the curve. In 6 patients who were dose-capped to 3750 IU for body mass index (BMI) ≥ 40, all exhibited therapeutic SAA without any asparaginase-related toxicity. Conclusions: To our knowledge this is the first single-institution post-market retrospective quality and safety analysis comparing CalPEG and PEGasp therapeutic drug monitoring and toxicity. Interestingly, the incidence of pancreatitis and VTE is significantly higher in patients receiving CalPEG and disproportionately higher in T-lineage. Median peak SAA levels were not predictive of toxicity, though GLM may help provide a predictive model. The rate of SI emphasizes the need for continued resource utilization of SAA monitoring. Our observed differences in CalPEG and PEGasp toxicity are discordant with two previously published clinical trials used for FDA approval of CALPEG (AALL07P4 and DFCI 11-001). Larger analyses of CalPEG SAA and toxicities are necessary to determine if these trends are reproducible. This information will help establish optimal utilization of SAA safety monitoring with the broader goal of identifying necessary practice changes in CalPEG clinical management.

The efficacy and toxicity of L-asparaginase in the treatment of acute lymphoblastic leukemia in children

L-asparaginase, an enzyme used as an anticancer drug, was one of the first drugs included in the treatment protocols for acute lymphoblastic leukemia. It has become widely used when an important metabolic feature of leukemia cells – their high demand for asparagine to maintain viability – was discovered. Three L-asparaginase preparations are currently used in clinical practice: native E. coli asparaginase, pegylated E. coli asparaginase (PEG-asparaginase), and native E. chrysanthemi-derived asparaginase, which have different half-lives, immunogenic profiles, and the spectrum and frequency of toxic effects. One of the main factors limiting the use of L-asparaginase is its high immunogenicity which can cause acute allergic reactions and the phenomenon of silent inactivation. The development of the immune response leads to an accelerated asparaginase clearance and a shortening of its half-life. To monitor the effectiveness of therapy with L-asparaginase, therapeutic drug monitoring of serum asparaginase activity can be used. When choosing management strategies for patients experiencing acute hypersensitivity reactions to L-asparaginase, the following factors should be taken into consideration: the severity of reaction, the number of previous exposures to L-asparaginase and serum asparaginase activity. The use of PEG-asparaginase is the best first-line treatment strategy for children acute lymphoblastic leukemia, its advantages include a significant reduction in the risk of developing acute allergic reactions, higher therapeutic efficacy and, as a result, improved treatment outcomes.

A Pilot Study Conducted at a Tertiary Cancer Care Center, Evaluating the Serum Asparaginase Activity in Children Suffering from Acute Lymphoblastic Leukemia after the Administration of Biosimilar Pegaspargase

Abstract Introduction L-asparaginase is considered to be the most important component in the treatment of acute lymphoblastic leukemia (ALL). Intensifying the use of L-asparaginase during treatment for ALL has resulted in a significant rise in the percentage of children and adolescents who are cured of the disease. Asparaginase trough activity more than or equal to 100 IU/L on day 7 has been found to be the desired activity level in all childhood leukemia patients. Objectives Due to the paucity of data on biosimilar pegaspargase in the upfront setting, we planned this prospective pilot study to evaluate the levels of serum asparaginase activity (SAA) after biosimilar pegaspargase infusion. Materials and Methods It is a prospective, single-center, pilot study of 10 pediatric ALL patients for the duration of 6 months. All children less than 18 years with ALL on treatment with curative intent and receiving pegaspargase and who provided informed consent were included in this study. The enzymatic spectrophotometric method was used to determine SAA, and it was measured on the 7th and 14th days after the first dosage of pegaspargase-asparaginase, as well as on the 14th day after the second dose of pegaspargase-asparaginase, while toxicity was charted according to Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Results From 10 patients with a median age of 5.5 years, a grand total of 29 samples were taken for analysis. Children who received pegaspargase had either B-ALL or T-ALL. After the first dose, mean ± SD (standard deviation), SAA levels at day 7 was 131.3 ± 38 IU/L and at Day 14 was 94.8 ± 8 IU/L. After the second dose, mean ± SD SAA level at day 14 was 86.1 ± 15 IU/L. No patient had clinical hypersensitivity reaction and no patient reported any asparaginase-related toxicity. One patient died due to sepsis, infection with multidrug-resistant gram-negative bacteria. Conclusions Biosimilar pegaspargase maintained good SAA levels 7 and 14 days after infusion. Drug Trial Registration: Clinical Trial Registry of India vide reference CTRI/2021/08/036033 and available at https://ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=59285&amp;EncHid=&amp;userName=

Molecular Modeling and Optimization of Type II E.coli l-Asparginase Activity by in silico Design and in vitro Site-directed Mutagenesis.

L-asparaginase (also known as L-ASNase) is a crucial therapeutic enzyme that is widely used in treatment of ALL (acute lymphoblastic leukemia) as a chemotherapeutic drug. Besides, this enzyme is used in the food industry as a food processing reagent to reduce the content of acrylamide in addition to the clinical industry. The improvement of activity and kinetic parameters of the L-ASNase enzyme may lead to higher efficiency resulting in practical achievement. In order to achieve this goal, we chosen glycine residue in position 88 as a potential mutation with advantageous outcomes. In this study, firstly to find the appropriate mutation on glycine 88, various in silico analyses, such as MD simulation and molecular docking, were carried out. Then, the rational design was adopted as the best strategy for molecular modifications of the enzyme to improve its enzymatic properties. Our in silico findings show that the four mutations G88Q, G88L, G88K, and G88A may be able to increase L-ASNase's asparaginase activity. The catalytic efficiency of each enzyme (kcat/Km) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the kcat/Km for the G88Q mutant is 36.32% higher than the Escherichia coli K12 ASNase II (wild type), which suggests that L-ASNase activity is improved at lower concentration of L-ASN. Kinetic characterization of the mutants L-ASNase activity confirmed the high turnover rate (kcat) with ASN as substrate relative to the wild type enzyme. In silico analyses and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.