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Background: Asparaginase is essential for treating T‐cell acute lymphoblastic leukemia (T‐ALL). Despite the ongoing debate on whether T‐ALL and T‐cell lymphoblastic lymphoma (T‐LBL) are the same disease entity or two distinct diseases, patients with T‐LBL often receive the same or similar treatment protocols as those with T‐ALL. Methods: The outcomes of patients with or without L‐asparaginase discontinuation were retrospectively analyzed among four national protocols: Japan Association of Childhood Leukemia Study (JACLS) ALL‐02 and ALL‐97 for T‐ALL and Japanese Pediatric Leukemia/Lymphoma Study Group ALB‐NHL03 and JACLS NHL‐98 for T‐LBL. The hazard ratio (HR) was calculated with the Cox regression model by considering L‐asparaginase discontinuation as a time‐dependent variable. Results: In total, 199 patients with T‐ALL, and 133 patients with T‐LBL were included. L‐asparaginase discontinuation compromised event‐free survival (EFS) of T‐ALL patients (ALL‐02: HR 3.32, 95% confidence interval [CI] 1.40–7.90; ALL‐97: HR 3.39, 95%CI 1.19–9.67). Conversely, EFS compromise was not detected among T‐LBL patients (ALB‐NHL03: HR 1.39, 95%CI 0.41–4.68; NHL‐98: HR 0.92, 95%CI 0.11–7.60). Conclusion: The effects of L‐asparaginase discontinuation differed between T‐ALL and T‐LBL. We assume that the differential impact results from (1) the inherent differential response to L‐asparaginase between them and/or (2) a less stringent assessment of early treatment response in T‐LBL than in T‐ALL. Given the poor salvage rate of refractory or relapsed T‐ALL and T‐LBL, optimization of the frontline therapy is critical, and the current study provides a new suggestion for further treatment modifications. However, larger studies in contemporary intensified treatment protocols are required. [ABSTRACT FROM AUTHOR]

This article discusses the case of a 4-year-old boy with severe hemophilia A who was receiving prophylactic treatment with emicizumab and developed acute lymphoblastic leukemia. The article describes how the child was treated and monitored during chemotherapy while continuing emicizumab. The authors highlight the concern of drug interactions, particularly between asparaginase and emicizumab. The study found that the combination of PEG-asparaginase with emicizumab did not affect the efficacy of emicizumab, and no bleeding complications occurred. The authors emphasize the need for close monitoring of hemostasis during chemotherapy in patients receiving emicizumab prophylaxis. [Extracted from the article]

Paediatric regimens which incorporate asparaginase have improved outcomes of adolescents and younger adults with acute lymphoblastic leukaemia but are limited by toxicity. This retrospective report by Tinajero and Xu suggests that delaying the timing of asparaginase administration during ALL induction may reduce risk of hepatotoxicity.Commentary on: Tinajero et al. Delaying pegaspargase during induction in adults with acute lymphoblastic leukaemia is associated with lower risk of high‐grade hepatotoxicity without adversely impacting outcomes. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19880. [ABSTRACT FROM AUTHOR]

The early detection of anti‐asparaginase biomarker can facilitate timely modification of asparaginase chemotherapy, thereby avoiding serious complications. Herein we describe the preparation of a novel electrochemical biosensing interface for rapid detection of anti‐asparaginase in the picomolar range (1–10 000 pM). Coimmobilization of ferrocene and asparaginase on a carbon interface (via diazonium grafting) facilitates transduction through attenuation of the surface‐bound ferrocene redox couple. The limit of detection of 0.8 pM for this point‐of‐care applicable method compares favourably to that of traditional faradaic assaying (2.0 pM) where transduction occurs by the target blocking the diffusion of the solution redox probe [Fe(CN)6]3−/4−. [ABSTRACT FROM AUTHOR]

Summary: Asparaginase is an essential drug for acute lymphoblastic leukaemia (ALL) treatment, but has several side effects, and its discontinuation often compromises patient outcomes. In the prospective Japan Association of Childhood Leukaemia Study ALL‐02 protocol, two major changes were made: (1) additional chemotherapies to compensate for the reduction of treatment intensity when asparaginase was discontinued and (2) more intensive concomitant corticosteroid administration, relative to our previous ALL‐97 protocol. In ALL‐02 study, 1192 patients were included and L‐asparaginase was discontinued for 88 (7.4%). Discontinuation due to allergy was markedly decreased relative to the ALL‐97 protocol (2.3% vs 15.4%). Event‐free survival (EFS) among patients with T‐ALL was compromised when L‐asparaginase was discontinued, as well as among patients with high‐risk B‐cell ALL, especially when discontinued before maintenance therapy. Moreover, multivariate analysis identified discontinuation of L‐asparaginase as an independent poor prognostic factor for EFS. In the current study, additional chemotherapies failed to fully compensate for L‐asparaginase discontinuation, illustrating the difficulty of replacing asparaginase with other classes of drugs, although this study was not designed to evaluate the effect of these modifications. Concomitant intensive corticosteroid treatment may help to reduce allergy to asparaginase. These results will assist in further optimization of asparaginase use. [ABSTRACT FROM AUTHOR]

Summary: Venous thromboembolism (VTE) is a well‐known complication in patients with acute lymphoblastic leukaemia (ALL) receiving asparaginase (ASP)‐based chemotherapy, including the ASP‐intensive Dana‐Farber Cancer Institute (DFCI) 91–01 protocol for adults. Since 2019, native L‐ASP is no longer available in Canada and was replaced by pegylated (PEG)‐ASP. To determine whether the incidence of VTE has changed since switching from L‐ASP to PEG‐ASP, we conducted a single‐centred retrospective cohort study. We included 245 adult patients with Philadelphia chromosome negative ALL between 2011 and 2021, with 175 from the L‐ASP group (2011–2019) and 70 from the PEG‐ASP group (2018–2021). During Induction, 10.29% (18/175) of patients who received L‐ASP developed VTE, whereas 28.57% (20/70) of patients who received PEG‐ASP developed VTE (p = 0.0035; odds ratio [OR] 3.35, 95% confidence interval [CI] 1.51–7.39), after adjusting for line type, gender, history of VTE, platelets at diagnosis. Similarly, during Intensification, 13.64% (18/132) of patients had VTE on L‐ASP while 34.37% (11/32) of patients on PEG‐ASP developed VTE (p = 0.0096; OR 3.96, 95% CI 1.57–9.96 with multivariable analysis). We found that PEG‐ASP is associated with a higher incidence of VTE compared to L‐ASP, both during Induction and Intensification, despite the administration of prophylactic anticoagulation. Further VTE mitigation strategies are needed in particular for adult patients with ALL receiving PEG‐ASP. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine—the selection medium for Gs‐based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co‐transfection of Gs‐Enbrel and Aspg‐Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5‐fold higher specific productivity and ~3‐fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics. [ABSTRACT FROM AUTHOR]

l‐asparaginases from mesophilic bacteria (ASNases), including two enzymes very successfully used in the treatment of leukaemia, have been consistently described as homotetramers. On the contrary, structural studies show that homodimers of these enzymes should be sufficient to carry out the catalytic reaction. In this report, we investigated whether the type I Yersinia pestis asparaginase (YpAI) is active in a dimeric form or whether the tetrameric quaternary structure is critical for its activity. Using multiple biophysical techniques that investigate enzymatic properties and quaternary structure at either high or low protein concentration, we found that dimeric YpAI is fully active, suggesting that the tetrameric form of this subfamily of enzymes does not bear significant enzymatic relevance. In this process, we extensively characterized YpAI, showing that it is a cooperative enzyme, although the mechanism of allostery is still not definitely established. We showed that, like most type I ASNases, the substrate affinity of YpAI is low and this enzyme is very similar in terms of both the structure and enzymatic properties to homologous type I ASNase from Escherichia coli (EcAI). We extended these studies to more medically relevant type II ASNases, used as anti‐leukaemia drugs. We confirmed that type II ASNases are not allosteric, and that they might also be functional in a dimeric form. However, the determination of the accurate tetramer⇆dimer dissociation constants of these enzymes that most likely lie in the picomolar range is not possible with currently available biophysical techniques. [ABSTRACT FROM AUTHOR]

B Conclusions: b Through CRISPR/Cas9 genome-wide knockout screening and long-time low-dose asparaginase treatment and then sequencing comparing with parental cell lines, we found that SOS1 gene can significantly affect asparaginase sensitivity in NKTCL. B Introduction: b Extranodal nature killer/T cell lymphoma (NKTCL) accounts for about 6% of all kinds of lymphoma. [Extracted from the article]

The article presents the discussion on Acute lymphoblastic leukemia (ALL) being a heterogeneous group of lymphoid neoplasms. Topics include characterized by the monoclonal proliferation of lymphoblasts in the bone marrow, peripheral blood, and other organs; and randomized trials asparaginase-containing regimens demonstrating a survival benefit for pediatric patients.

Adults with relapsed or refractory B‐precursor acute lymphoblastic leukaemia (R/R BCP‐ALL) have very poor outcome. Blinatumomab as single agent has shown activity in R/R BCP‐ALL. We aimed to assess the activity of blinatumomab in concomitant association with intensive chemotherapy. Seventeen patients with R/R BCP‐ALL were treated with combination of blinatumomab and VANDA (etoposide, cytarabine, mitoxantrone, dexamethasone and asparaginase) regimen. Complete remission (CR) was achieved in 14/17 patient (82%) and 11/17 (65%) were transplanted. One‐year leukaemia‐free survival was 58.8% for the whole cohort and 90.9% for transplanted patients. These preliminary data suggest that the VANDA‐blinatumomab salvage regimen leads to a very high rate of CR and HSCT in suitable patients. [ABSTRACT FROM AUTHOR]

Osteonecrosis (ON) is a complication of acute lymphoblastic leukaemia (ALL) treatment with patient‐ (age, female sex, genetic polymorphisms, presence of metabolic syndrome) and treatment‐specific (glucocorticoid type and schedule) risk factors described. The potential role of asparaginase in increasing risk of ON via effects on coagulation, lipid metabolism, and steroid clearance is now also recognised. Paediatric studies consistently identify age as a key risk factor for ON, with adolescents at higher risk than young children. Fewer studies comprehensively report on risk of ON in adults, but available evidence suggests that adolescents and young adults (AYAs) treated with corticosteroid and asparaginase‐containing paediatric‐inspired regimens are more at risk than older adults treated with paediatric‐inspired or traditional adult regimens. There are few proven strategies to prevent or mitigate the severity of ON and other orthopaedic complications of ALL therapy. Future clinical trials should carefully ascertain orthopaedic adverse events in adults. Evidence‐based guidelines should be developed for management of orthopaedic adverse events in adults being treated for ALL, especially high‐risk AYAs being treated with paediatric‐inspired regimens. [ABSTRACT FROM AUTHOR]

Summary: Asparaginase is essential in treating acute lymphoblastic leukaemia (ALL). Asparaginase‐related hypersensitivity causes treatment discontinuation, which is associated with decreased event‐free survival. To continue asparaginase treatment after hypersensitivity, a formulation of asparaginase encapsulated in erythrocytes (eryaspase) was developed. In NOR‐GRASPALL 2016 (NCT03267030) the safety and efficacy of eryaspase was evaluated in 55 patients (aged 1–45 years; median: 6.1 years) with non‐high‐risk ALL and hypersensitivity to asparaginase conjugated with polyethylene glycol (PEG‐asparaginase). Eryaspase (150 u/kg) was scheduled to complete the intended course of asparaginase (1–7 doses) in two Nordic/Baltic treatment protocols. Forty‐nine (96.1%) patients had asparaginase enzyme activity (AEA) ≥100 iu/l 14 ± 2 days after the first eryaspase infusion [median AEA 511 iu/l; interquartile range (IQR), 291–780], whereas six of nine (66.7%) patients had AEA ≥100 iu/l 14 ± 2 days after the fourth infusion (median AEA 932 iu/l; IQR, 496–163). The mean terminal half‐life of eryaspase following the first infusion was 15.3 ± 15.5 days. Few asparaginase‐related adverse events were reported; five patients (9.1%) developed clinical allergy associated with enzyme inactivation. Replacement therapy was successfully completed in 50 patients (90.9%). Eryaspase was well tolerated, and most patients had AEA levels above the therapeutic target after the first infusion. The half‐life of eryaspase confirmed that a 2‐week schedule is appropriate. [ABSTRACT FROM AUTHOR]

BACKGROUND: The treatment of acute lymphoblastic leukemia (ALL) uses the biopharmaceutical l‐asparaginase (ASNase) as the main medication. This drug, from bacterial origin (Escherichia coli or Erwinia chrysanthemi), depletes l‐asparagine (Asn) and secondarily l‐glutamine (Gln – GLNase activity) from the bloodstream, leading leukemic cells to die by deprivation of Asn. The use of ASNase is limited by the high incidence of adverse effects, which collectively can specifically impair quality of life of patients. Its high toxicity caused by the product of the hydrolysis of amino acids and the formation of anti‐ASNase antibodies often required treatment interruption, thus reducing the chances of cure and increasing the rates of disease relapse. RESULTS: In order to improve enzymatic activity, while reducing toxicity, we developed through directed evolution a double‐mutant ASNase from Erwinia chrysanthemi (Erw_DM), which has specific activity for Asn 46% higher than the wild‐type enzyme (Erw_WT). This makes it possible to reduce the amount of protein necessary for depletion of this amino acid and, consequently, the reduction of GLNase activity, considered toxic. In silico analysis showed that a lower number of epitopes was exposed, resulting in reduced recognition of the recombinant protein by antibody anti‐ASNase observed in vitro assay. Furthermore, we observed the same cytotoxic profile for the MOLT‐4 and REH ALL cell lines using 40% lower protein mass of Erw_DM to achieve the minimum enzyme activity required in the bloodstream during treatment. CONCLUSION: Altogether, our findings describe a potent and less immunogenic ASNase, an improvement that may alleviate treatment adverse effects developed in anti‐ALL therapy. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

JZP‐458 is a recombinant Erwinia asparaginase produced using a novel Pseudomonas fluorescens expression platform that yields an enzyme expected to lack immunologic cross‐reactivity to Escherichia coli–derived asparaginases. It is being developed as part of a multiagent chemotherapeutic regimen to treat acute lymphoblastic leukemia or lymphoblastic lymphoma patients who develop E coli–derived asparaginase hypersensitivity. A population pharmacokinetic (PopPK) model was developed for JZP‐458 using serum asparaginase activity (SAA) data from a phase 1, single‐dose study (JZP458‐101) in healthy adults. Effects of intrinsic covariates (body weight, body surface area, age, sex, and race) on JZP‐458 PK were evaluated. The model included SAA data from 24 healthy adult participants from the phase 1 study who received JZP‐458: intramuscular (IM) data at 12.5 mg/m2 (N = 6) and 25 mg/m2 (N = 6), and intravenous (IV) data at 25 mg/m2 (N = 6) and 37.5 mg/m2 (N = 6). Model simulations of adult and pediatric SAA profiles were performed to explore the likelihood of achieving a therapeutic target nadir SAA (NSAA) level ≥0.1 IU/mL based on different administration strategies. PopPK modeling and simulation suggest JZP‐458 is expected to achieve 72‐hour NSAA levels ≥0.1 IU/mL in 100% of adult or pediatric populations receiving IM administration at 25 mg/m2, and in 80.9% of adult and 94.5% of pediatric populations receiving IV administration at 37.5 mg/m2 on a Monday/Wednesday/Friday (M/W/F) dosing schedule. Based on these results, the recommended starting dose for the phase 2/3 pivotal study is 25 mg/m2 IM or 37.5 mg/m2 IV on a M/W/F dosing schedule in pediatric and adult patients. [ABSTRACT FROM AUTHOR]

Asparaginase (ASP) is an essential component for the acute lymphoblastic leukemia (ALL) treatment, but toxicities, such as allergy, frequently limit its use. Although the potentially lower PEG‐ASP formulation immunogenicity, few studies with conflicting results have compared the allergy incidence between Escherichia coli‐ASP and PEG‐ASP in the same protocol. We aimed at comparing the allergy incidence in children receiving native E. coli‐ASP versus PEG‐ASP within the same clinical protocol (Spanish Society of Pediatric Hematology and Oncology ALL‐SEHOP‐PETHEMA 2013). One hundred and twenty‐six children (1–19 years) diagnosed with ALL from 2013 to 2020 were included. Patients in group 1 received a sequential scheme of native E. coli‐ASP 10,000 IU/m2 intramuscularly (IM) followed by PEG‐ASP 1000 IU/m2 IM. Patients in group 2 received PEG‐ASP 1000 IU/m2 IM upfront. Clinical allergy incidence was compared between both groups. Serum ASP activity (SAA) was measured in a subgroup of patients, and silent inactivation was recorded. The cumulative incidence of clinical allergy was significantly higher in group 1 (native followed by PEG‐ASP) than in group 2 (PEG‐ASP upfront), 24.7% versus 4.1% (p = 0.0085). Adequate ASP activity was achieved with PEG‐ASP 1000 IU/m2 dose in most patients (median SAA 412.5 and 453.0 IU/L at days 7 and 14). The incidence of silent inactivation in PEG‐ASP upfront patients was very low. PEG‐ASP‐used upfront was associated with a lower incidence of clinical allergy than that observed in the sequential use of native E. coli‐ASP followed by PEG‐ASP. PEG‐ASP at 1000 IU/m2 was effective in achieving enough ASP activity in most patients. [ABSTRACT FROM AUTHOR]

Background: Pegaspargase (PEG‐ASP) is an integral component of therapy for acute lymphoblastic leukemia (ALL) but is associated with hepatotoxicity that may delay or limit future therapy. Obese and adolescent and young adult (AYA) patients are at high risk. Levocarnitine has been described as potentially beneficial for the treatment or prevention of PEG‐ASP‐associated hepatotoxicity. Methods: We collected data for patients age ≥10 years who received levocarnitine during induction therapy for ALL, compared to a similar patient cohort who did not receive levocarnitine. The primary endpoint was conjugated bilirubin (c.bili) >3 mg/dl. Secondary endpoints were transaminases >10× the upper limit of normal and any Grade ≥3 hepatotoxicity. Results: Fifty‐two patients received levocarnitine for prophylaxis (n = 29) or rescue (n = 32) of hepatotoxicity. Compared to 109 patients without levocarnitine, more patients receiving levocarnitine were obese and/or older and had significantly higher values for some hepatotoxicity markers at diagnosis and after PEG‐ASP. Levocarnitine regimens varied widely; no adverse effects of levocarnitine were identified. Obesity and AYA status were associated with an increased risk of conjugated hyperbilirubinemia and severe transaminitis. Multivariable analysis identified a protective effect of levocarnitine on the development of c.bili >3 mg/dl (OR 0.12, p = 0.029). There was no difference between groups in CTCAE Grade ≥3 hepatotoxicity. C.bili >3 mg/dl during induction was associated with lower event‐free survival. Conclusions: This real‐world data on levocarnitine supplementation during ALL induction highlights the risk of PEG‐ASP‐associated hepatotoxicity in obese and AYA patients, and hepatotoxicity's potential impact on survival. Levocarnitine supplementation may be protective, but prospective studies are needed to confirm these findings. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cell‐free protein synthesis (CFPS) technology has emerged as a powerful tool for a variety of biotechnological applications, including the expression of different classes of biopharmaceutical products. L‐Asparaginase (E.C. Number: 3.5.1.1, L‐asparagine amidohydrolase) (L‐ASNase) is an important biopharmaceutical used to treat leukemia, but expression of multiple proteoforms in CFPS systems and rapid characterization using standard colorimetric methods has not yet been fully exploited. Herein, recombinant expression and characterization of an L‐ASNase from Erwinia chrysanthemi (Erwinase) using a new CFPS protocol is reported. RESULTS: Expression and quantification of the enzymatic activity of a soluble his‐tagged L‐ASNase directly from a CFPS reaction was successfully achieved. Purification of the protein was not required in order to assess its biological activity. Activity of L‐ASNase was significantly higher than the control reaction (7.07 ± 0.68 U mL–1vs. 1.83 ± 0.14 U mL–1, respectively). Expression of a mutant Erwinase proteoform – V293M – was also achieved and it presented a similar enzymatic activity. No significant loss in L‐ASNase enzymatic activity was noticed after removal of cyclic AMP, spermidine, transfer RNA, T7 RNA polymerase and, especially, ammonium acetate (a common interference in ASNase enzymatic assays) from the CFPS reaction. CONCLUSION: The protocol developed in this work will facilitate the screening of novel clinically‐relevant L‐ASNase proteoforms. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Inherited GATA3 variant associated with positive minimal residual disease in childhood B-cell acute lymphoblastic leukemia via asparaginase resistance We genotyped I GATA3 i SNPs rs3824662 and rs3781093 in 308 children with B-ALL enrolled in the CCCG-ALL-2015 study to evaluate their association with ALL treatment outcomes in the Han Chinese population (Figure 1A, Table S1). Dear Editor, Genome-wide association studies have identified that germline single nucleotide polymorphisms (SNPs) in I GATA3 i significantly influence the treatment outcomes of childhood acute lymphoblastic leukemia (ALL).1-3 However, the role of inherited I GATA3 i variants in Han Chinese patients with B-cell ALL (B-ALL) and the molecular mechanisms by which these variants are linked to poor prognosis are largely unknown. Surprisingly, the rs3824662 A risk allele significantly increased the enhancer activity by approximately threefold compared to the nonrisk allele in GM18900, Nalm6, and Reh cells, while the rs3781093 C risk allele did not affect I GATA3 i transcription (Figure 2C, Figure S4). [Extracted from the article]

Summary: Our group recently showed that the (ASNase) formulation available in Brazil from 2017 to 2018 when used at the same dose and frequency as the formulation provided previously did not reach the activity considered therapeutic. Based on these, our goal was to assess the impact of these facts on the prognosis of children with ALL at different oncology centers. A multicentre retrospective observational study followed by a prospective follow‐up. Patients aged >1 and <18 years in first‐line treatment followed up at 10 referral centres, between 2014 and 2018 who received the formulation Leuginase® were identified (Group B). For each patient, the centre registered 2 patients who received ASNase in the presentation of Aginasa® exclusively (Group A). Data collection was registered using (Redcap®). A total of 419 patients were included; 282 in Group A and 137 in B. Group A had a 3‐year OS and EFS of 91·8% and 84·8% respectively, while Group B had a 3‐year OS of 83·8% (P = 0·003) and EFS of 76·1% (P = 0·008). There was an impact on 3‐year OS and EFS of children who received a formulation. This result highlights the importance of evaluating ASNase and monitoring its activity. [ABSTRACT FROM AUTHOR]

Asparaginase (Asp) is one of the most important drugs for treating acute lymphoblastic leukemia (ALL). However, off‐protocol Asp administration (OPAA) or hypersensitivity may disturb its pharmacokinetic profile. In this retrospective study, we sought to determine whether OPAA and hypersensitivity to Escherichia coli asparaginase (E coli Asp) impaired extramedullary relapse prevention in a pediatric ALL cohort treated according to SCMC‐ALL‐2005 protocol from 2005 to 2014 at the Shanghai Children's Medical Center (SCMC). In total, 676 patients were enrolled in this study, including 369 with OPAA and 60 exhibiting hypersensitivity to E coli Asp. At the end of the most recent follow‐up, 58 patients had extramedullary relapse. The 5‐year cumulative extramedullary relapse incidence in patients with OPAA was 11.01%, whereas that in patients without OPAA was 5.28% (P =.0036). Moreover, the 5‐year cumulative extramedullary relapse incidence in patients that exhibited hypersensitivity to E coli Asp was 16.48%, whereas that in patients without hypersensitivity was 7.59% (P =.0195). Concerning the relapse site, OPAA not only increased central nervous system (CNS) relapse but testicular relapse as well. Based on Fine and Gray multivariate analysis, OPAA and hypersensitivity to Asp were independent risk factors for extramedullary relapse. In conclusion, to prevent extramedullary relapse of ALL, adequate duration to administrate Asp was more important than the total dosage, and more attention should be paid to Asp inadequate due to hypersensitivity. What's new? Asparaginase is one of the most important drugs for treating acute lymphoblastic leukemia. However, off‐protocol asparaginase administration or hypersensitivity may disturb its pharmacokinetic profile. This is the first retrospective study to demonstrate that off‐protocol reduction of injection times during a chemotherapy course with no change in the total asparaginase dosage could significantly increase the risk of extramedullary relapse, including central nervous system and testicular relapse. A higher cumulative extramedullary relapse incidence was also observed in patients hypersensitive to asparaginase. Asparaginase is important for ameliorating overall survival and preventing extramedullary relapse, and attention should be paid to optimising administration. [ABSTRACT FROM AUTHOR]

Aims: Asparaginase (ASP) hypersensitivity is a well‐known challenge in the treatment of lymphoblastic malignancies. In terms of cost considerations, the cheap native Escherichia coli ASP, the most immunogenic form of this medication, is used in the first line in middle‐income countries. Previously, the role of the HLA‐DRB1*07:01–DQA1*02:01–DQB1*02:02 haplotype had been established to associate with E. coli ASP hypersensitivity. We investigated a possible cost‐effective genetic testing method to identify patients harbouring the risk HLA haplotype in order to pave the way for safer ASP treatment. Methods: In 241 patients with previously determined HLA‐DRB1*07:01–DQA1*02:01–DQB1*02:02 haplotype and known ASP hypersensitivity status, 4 candidate HLA‐tagging single‐nucleotide polymorphisms (SNP)s were measured, and the performance of the different sets of these tag SNPs was evaluated. Results: We identified a combination of 2 SNPs — rs28383172 and rs7775228 — as a tag for HLA‐DRB1*07:01–DQA1*02:01–DQB1*02:02 haplotype with sensitivity and specificity values >95%. In line with previous findings, we found complete concordance between HLA‐DRB1*07:01 and rs28383172. With bioinformatics methods, the results were also confirmed in the 1000 Genomes dataset in different ethnic groups. Conclusion: Rs28383172 and rs7775228 are suitable for identifying HLA‐DRB1*07:01–DQA1*02:01–DQB1*02:02 carriers. Compared to the rest of the population, patients with hypersensitivity‐prone genotype would benefit more from the administration of less immunogenic PEGylated ASP before the hypersensitivity evolves, incurring minimal extra cost. [ABSTRACT FROM AUTHOR]

L‐asparaginase has been an important component of acute lymphoblastic leukemia (ALL) therapy for over 40 years, and is standard therapy during ALL induction and consolidation treatment. L‐asparaginases are immunogenic and can induce hypersensitivity reactions; inability to receive asparaginase has been associated with poor patient outcomes. There are L‐asparaginases of varied bacterial origins, with the most commonly used being Escherichia coli (E. coli); therefore, to ensure that patients who develop hypersensitivity to E. coli‐derived asparaginases receive an adequate therapeutic course, alternative preparations are warranted. JZP‐458 is a recombinant Erwinia asparaginase produced using a novel Pseudomonas fluorescens expression platform that yields an enzyme with no immunologic cross‐reactivity to E. coli‐derived asparaginases. To evaluate the safety, tolerability, and pharmacokinetics (PK) of a single dose of JZP‐458, a randomized, single‐center, open‐label, phase I study was conducted with JZP‐458 given via i.m. injection or i.v. infusion to healthy adult volunteers. At the highest doses tested for each route of administration (i.e., 25 mg/m2 i.m. and 37.5 mg/m2 i.v.), JZP‐458 achieved serum asparaginase activity (SAA) levels ≥ 0.1 IU/mL at 72 hours postdose for 100% of volunteers. Bioavailability for i.m. JZP‐458 was estimated at 36.8% based on SAA data. All dose levels were well‐tolerated, with no unanticipated adverse events (AEs), no serious AEs, and no grade 3 or higher AEs. Based on PK and safety data, the recommended JZP‐458 starting dose for the pivotal phase II/III study in adult and pediatric patients is 25 mg/m2 i.m. and 37.5 mg/m2 i.v. on a Monday/Wednesday/Friday dosing schedule. [ABSTRACT FROM AUTHOR]

Prophylaxis with low-molecular-weight heparin reduces thrombotic events and allows continuation of asparaginase containing regimens during intensification phase Given our results, patients who received enoxaparin had a significantly lower rate of thrombosis compared to the control group without prophylaxis [ I p i = 0.01; odds ratio (OR) 0.42, 95% confidence interval (CI) 0.21-0.83]. Does anticoagulation prophylaxis reduce the venous thromboembolism rate in adult acute lymphoblastic leukaemia treated with asparaginase-based therapy?. [Extracted from the article]

BACKGROUND l‐asparaginase (ASNase), a biopharmaceutical enzyme used in the treatment of childhood lymphoid malignancies, is commercially produced from Escherichia coli and Erwinia chrysanthemi. However, it causes severe adverse effects due to allergenic prokaryotic epitopes on the protein surface. ASNase II from Saccharomyces cerevisiae can be a promising alternative source of this enzyme. In this study, conditions to produce ASNase from S. cerevisiae expressed in Pichia pastoris have been investigated in shake flasks and 3 L‐bioreactor. We evaluated if medium composition, concentration of carbon source (i.e. glycerol), growth time, concentration of inducer (i.e. methanol), temperature and initial pH influenced both biomass and ASNase expression. RESULTS: Biomass of around 53 g L–1 and ASNase volumetric activity of 710 U L–1 were achieved using the buffered glycerol‐complex medium (BMGY) containing 40 g L–1 glycerol, with induction after 141 h using 3.0% (v/v) methanol, at 20 °C and initial pH 6.0. CONCLUSION: The experiments performed in shake flasks were scalable to a 3 L‐bioreactor, suggesting that this bioprocess could be scaled‐up for industrial production. © 2020 Society of Chemical Industry (SCI) [ABSTRACT FROM AUTHOR]

Inspired by the amino acid composition of natural protein surfaces, we developed a zwitterionic cloak containing multi‐layers of short alternating glutamic acid and lysine (EK) peptides as a facile, highly effective and low‐immunogenicity approach for the protection and delivery of biotherapeutics. Each EK layer grafted to proteins provides multiple times of new lysine reaction sites for the growth of subsequent EK layers. This unique design allows EK peptides to achieve high coating density on proteins, overcoming the limitation of traditional conjugation strategies that rely on the number of innate lysine groups. A triple‐layer EK cloak manifests to successfully eliminate the specific and non‐specific interactions of protected asparaginase with biological media while prolong the drug circulation time and significantly mitigate its immunogenicity in vivo, suggesting an EK peptide cloak as a promising approach to improve the safety and efficacy of biotherapeutics. [ABSTRACT FROM AUTHOR]

Summary: Venous thromboembolism (VTE) is a well‐known complication in adults receiving asparaginase (ASNase)‐based intensification chemotherapy for acute lymphoblastic leukaemia (ALL). The optimal preventative strategy is unclear. Our objective is to determine the effects of low‐molecular‐weight heparin (LMWH) as primary VTE prophylaxis. A single‐centred retrospective cohort study of adult patients with Philadelphia chromosome negative (Ph−) ALL who received ASNase‐based intensification from 2001 to 2017, with prophylaxis given from 2011 to 2017. In all, 214 patients were included in this study with 99 in the historical control group and 125 in the prophylaxis group. The mean (range) enoxaparin dose was 0·79 (0·39–1·2) mg/kg. Of the 125 patients in the prophylaxis group 17 (13·6%) developed VTE during the intensification phase, while 27/99 patients (27·3%) in the control cohort experienced at least one thrombotic event (odds ratio [OR] 0·42, 95% confidence interval [CI] 0·21–0·83). Overall, the main sites of VTE incidences included deep vein thrombosis in the lower extremity (54·6%), pulmonary embolism (13·6%) and catheter‐related thrombosis (22·7%). In addition, we found that after adjusting for age, T‐phenotype ALL was associated with VTE development (OR 3·07, 95% CI 1·04–9·08). There was no documented major bleeding in the prophylaxis group. LMWH prophylaxis reduced the incidence of symptomatic VTE in adult patients with ALL receiving intensification chemotherapy with ASNase. [ABSTRACT FROM AUTHOR]

Summary: Asparaginase (ASNase) is an important anti‐leukaemic drug in the treatment of childhood acute lymphoblastic leukaemia (ALL) and non‐Hodgkin lymphoma (NHL). A substantial proportion of patients develop hypersensitivity reactions with anti‐ASNase neutralising antibodies, resulting in allergic reactions or silent inactivation (SI), and characterised by inactivation and rapid clearance of ASNase. We report results of a prospective, real‐time therapeutic drug monitoring of pegylated Escherichia coli (PEG‐)ASNase and Erwinia ASNase in children treated for ALL and NHL in Belgium. Erwinia ASNase was given as second‐line after hypersensitivity to PEG‐ASNase. In total, 286 children were enrolled in the PEG‐ASNase cohort. Allergy was seen in 11·2% and SI in 5·2% of patients. Of the 42 patients treated with Erwinia ASNase, 7·1% experienced allergy and 2·4% SI. The median trough PEG‐ASNase activity was high in all patients without hypersensitivity. After Erwinia administration significantly more day 3 samples had activities <100 IU/l (62·5% vs. 10% at day 2 (D2)). The median D2 activity was significantly higher for intramuscular (IM; 347 IU/l) than for intravenous Erwinia administrations (159 IU/l). This prospective, multicentre study shows that monitoring of ASNase activity during treatment of children with ALL and NHL is feasible and informative. Treatment with Erwinia ASNase warrants close monitoring and optimally adherence to a 2‐day interval of IM administrations. [ABSTRACT FROM AUTHOR]

Aims: L‐asparaginase is an essential medicine in the treatment of pediatric acute lymphoblastic leukemia (ALL) and the quality of generic formulations is an area of concern. We compared nine generic formulations of L‐asparaginase available in India with the innovator. Methods: The quality of formulations was assessed by measuring 72‐hour trough asparaginase activity in children with ALL during induction following administration of 10,000 IU/m2 of L‐asparaginase. In‐vitro analysis of the label claim was assessed by measuring activity of three generic formulations. Liquid chromatography‐mass spectrometry (LC/MS) was used to determine the amount of host contaminant proteins (HCPs) in the formulations. Results: Between March 2015 to June 2018, 240 samples from 195 patients were analyzed. The number of samples analyzed ranged from 7–66 per generic brand (median: 18) and seven of the innovator. The proportion of generic formulations that failed to achieve a predefined clinical threshold activity of 50 IU/L ranged from 16.7% (2/12) to 84.9% (28/33) in the highest activity to lowest activity generic respectively. On other hand, all innovator samples had activity greater than 50 IU/L. In‐vitro asparaginase activity in the three generic formulations tested ranged from 71.4–74.6% of the label claim (10,000 IU) compared to 93.5% for the innovator. LC/MS analysis of generic 5 identified 25 HCPs with a relative peptide count of 27.1% of the total peptides. Conclusions: Generic formulations had lower asparaginase activity which raises serious clinical concerns regarding their quality. Until stringent regulatory enforcement improves the quality of these generics, dose adaptive strategies coupled with therapeutic drug monitoring need to be considered. [ABSTRACT FROM AUTHOR]

Summary: Polyethylene glycol (PEG) conjugated asparaginase (PEGasparaginase) is essential for treatment of paediatric acute lymphoblastic leukaemia. We developed an assay identifying antibodies against the PEG‐moiety, the linker and the drug itself in patients experiencing hypersensitivity reactions to PEGasparaginase. Eighteen patients treated according to the DCOG ALL‐11 protocol, with a neutralizing hypersensitivity reaction to PEGasparaginase to the first PEGasparaginase doses in induction (12 patients) or during intensification after interruption of several months (6 patients) were included. ELISA was used to measure antibodies, coating with the succinimidyl succinate linker conjugated to BSA, PEGfilgrastim and Escherichia coli asparaginase, and using hydrolysed PEGasparaginase and mPEG5,000 for competition. Anti‐PEG antibodies were detected in all patients (IgG 100%; IgM 67%) of whom 39% had anti‐PEG antibodies exclusively. Pre‐existing anti‐PEG antibodies were also detected in patients who not previously received a PEGylated therapeutic (58% IgG; 21% IgM). Antibodies against the SS‐linker were predominantly detected during induction (50% IgG; 42% IgM). Anti‐asparaginase antibodies were detected in only 11% during induction but 94% during intensification. In conclusion, anti‐PEG and anti‐SS‐linker antibodies predominantly play a role in the immunogenic response to PEGasparaginase during induction. Thus, switching to native E. coli asparaginase would be an option for adequate asparaginase treatment. [ABSTRACT FROM AUTHOR]

Asparaginase is fundamental to the treatment of haematological malignancies. However, little has been studied on the effects that asparaginase could exert on solid tumours. Thus, this study aimed to evaluate the effects of asparaginase on an oral carcinoma cell line. The cytotoxicity of asparaginase in SCC‐9 (tongue squamous cell carcinoma) and HaCaT (human keratinocyte) cell lines was evaluated with MTT cell viability assay. The cells were treated with asparaginase at 0.04, 0.16, 0.63, 1.0, 1.5, 2.5, and 5.0 IU/mL. Dose‐response curves and IC50 values were obtained and the Tumour Selectivity Index (TSI) was calculated. The effect of asparaginase on procaspase‐3 and nuclear factor κB (NFκB) expression was evaluated with western blot because it was reported that the overexpression of NFκB has been shown to contribute to tumour cell survival, proliferation, and migration. Caspase 3/7 staining was performed to identify cell death using flow cytometry. Effective asparaginase concentrations were lower for SCC‐9 cells when compared to HaCaT cells. The cytotoxicity results at 48 and 72 hours were significantly different for SCC‐9 cells. The TSI indicated that asparaginase was selective for the tumour cells. A decrease in procaspase‐3 and NFκB protein levels was observed in SCC‐9 cells. Furthermore, asparaginase resulted in significant apoptosis after 48 and 72 hours. Based on these results, asparaginase was cytotoxic in a dose‐ and time‐dependent manner, induces apoptosis, and reduces NFκB expression in oral cancer cells. These results encourage further studies on the effectiveness of this enzyme as a treatment for solid tumours, especially head and neck cancer. [ABSTRACT FROM AUTHOR]

This study used Korean national data to investigate the relationship between treatment patterns and outcomes in Korean adolescent and young‐adult (AYA) acute lymphoblastic leukaemia (ALL) patients. Chemotherapy incorporating L‐asparaginase was considered paediatric‐inspired (PI), as opposed to adult protocols. In total, 65·3% of patients received PI therapy. Five‐year overall survival (OS) of PI‐treated patients outperformed adult protocols (63·1% vs. 40·4%; P < 0·0001); this trend was maintained within various age subgroups. Younger age, L‐asparaginase therapy, and radiotherapy corresponded with superior OS by multivariable analysis. OS tends to improve with PI protocols that include L‐asparaginase in AYA ALL, suggesting that therapy protocol is critical in the treatment performance of this group. [ABSTRACT FROM AUTHOR]

Background: Rabacfosadine (RAB), a novel antineoplastic agent conditionally licensed for the treatment of lymphoma in dogs, is efficacious in both naïve and previously treated dogs. Its use in combination with L‐asparaginase (L‐ASP) has not been studied. Hypothesis/Objectives: To evaluate the safety and efficacy of L‐ASP given concurrently with RAB in dogs with relapsed multicentric lymphoma. Animals Fifty‐two dogs with relapse of lymphoma after treatment with at least 1 doxorubicin‐based chemotherapy protocol. Methods: Open‐label, multicenter, prospective single‐arm clinical trial. Dogs were treated with RAB at 1.0 mg/kg IV every 21 days for up to a total of 5 doses. L‐asparaginase was administered at 400 IU/kg SQ concurrently with the first 2 treatments of RAB. Results: The overall response rate (ORR) for all dogs was 67%, with 19 dogs (41%) achieving a complete response (CR). The median progression‐free survival time (MPFS) was 63 days (range 5‐428 days). Dogs experiencing a CR as their best response had an MPFS of 144 days (range 44‐428 days). Adverse events were similar to previous studies evaluating single agent RAB. Failure to achieve a CR and having previously received L‐ASP were negative prognostic factors on multivariate analysis. Conclusions and Clinical Importance: Concurrent RAB/L‐ASP appears to be both efficacious and safe for treating relapsed multicentric lymphoma in dogs. Adverse events were most often mild and no unexpected toxicoses were observed. [ABSTRACT FROM AUTHOR]

Summary: Outcomes in childhood T‐cell acute lymphoblastic leukaemia (T‐ALL) are steadily improving due to intensive therapy. Between 1989 and 2008, 599 children with newly diagnosed T‐ALL were enrolled in two successive European Organization for Research and Treatment of Cancer ‐ Children's Leukaemia Group trials (58881 and 58951), both based on the Berlin‐Frankfurt‐Munster protocol and without cranial irradiation. In the latter trial induction chemotherapy was intensified. The most important randomizations were Medac Escherichia coli asparaginase versus Erwinia asparaginase in trial 58881, and dexamethasone (6 mg/m2/day) versus prednisolone (60 mg/m2/day) and prolonged versus conventional asparaginase duration in trial 58951. 8‐year event‐free survival (EFS) increased from 65·1% to 74·0% in trial 58951. Improvement was most profound for patients with white blood cell (WBC) counts <100 × 109/l and "good responders" to prephase. Medac E. coli asparaginase was associated with longer EFS [hazard ratio (HR) 0·54, P = 0·0015] and overall survival (HR 0·51, P = 0·0018). Induction therapy with dexamethasone did not improve EFS compared to prednisolone. Remarkably, intensification of central nervous system (CNS)‐directed therapy in trial 58951 resulted in fewer bone marrow relapses, while the incidence of CNS relapses remained low. In summary, we showed that adequate asparaginase therapy, intensified induction treatment and intensification of CNS‐directed chemotherapy can result in an improvement of outcome in T‐ALL patients with good prephase response and initial WBC counts <100 × 109/l, representing approximately 50% of T‐ALL patients. [ABSTRACT FROM AUTHOR]

Summary: Eradicating the malignant stem cell is the ultimate challenge in the treatment of leukaemia. Leukaemic stem cells (LSC) hijack the normal haemopoietic niche, where they are mainly protected from cytotoxic drugs. The anti‐leukaemic effect of L‐asparaginase (ASNase) has been extensively investigated in acute lymphoblastic leukaemia, but only partially in acute myeloid leukaemia (AML). We explored the susceptibility of AML‐LSC to ASNase as well as the role of the two major cell types that constitute the bone marrow (BM) microenvironment, i.e., mesenchymal stromal cells (MSC) and monocytes/macrophages. Whilst ASNase was effective on both CD34+CD38+ and CD34+CD38− LSC fractions, MSC and monocytes/macrophages partially counteracted the effect of the drug. Indeed, the production of cathepsin B, a lysosomal cysteine protease, by BM monocytic cells and by AML cells classified as French‐American‐British M5 is related to the inactivation of ASNase. Our work demonstrates that, while MSC and monocytes/macrophages may provide a protective niche for AML cells, ASNase has a cytotoxic effect on AML blasts and, importantly, LSC subpopulations. Thus, these features should be considered in the design of future clinical studies aimed at testing ASNase efficacy in AML patients. [ABSTRACT FROM AUTHOR]

Adverse drug reactions (ADRs) are a major problem in modern medicine, representing up to the fourth‐highest cause of mortality. Pharmacogenomic tests are 1 of the most promising methods to tackle the challenge of ADRs. The objective of this study was to analyze the clinical and demographic information of the pan‐Canadian active surveillance network, Canadian Pharmacogenomics Network for Drug Safety (CPNDS). Information entered into the database by trained active surveillors between May 15, 2005 and May 9, 2017 was collected and analyzed. Specific data included for analysis were number of ADR reports, reports of drug use without ADRs, date of onset of ADR, suspected drugs, concomitant drugs, and fatal ADR cases. The CPNDS database consisted of 93,974 reports of medication use, including 10,475 reports of ADRs, of which 72.6% occurred in pediatric patients (≤21 years old). Self‐reported ancestries were predominantly Europe (38.2%), Canada (9.6%), and East Asia (4.9%). The 5 most frequent ADRs were cutaneous ADRs, peripheral neuropathy, cardiotoxicity, central nervous system toxicity, and ototoxicity. The 5 drugs most commonly suspected to cause ADRs were methotrexate, vincristine, doxorubicin, cisplatin, and L‐asparaginase. The CPNDS database is a valuable resource to identify clinical and genomic predictors of ADRs. The database also highlights our candidate ADRs for pharmacogenomic discovery research to identify additional ADR biomarkers. Additionally, the database provides information that can be used for developing strategies to prevent ADRs and raises awareness of ADRs among Canadian healthcare professionals. [ABSTRACT FROM AUTHOR]

l‐asparaginase has high application value in medicine and food industry, but the low yield limits its application. In this study, we designed a synthetic system in Bacillus subtilis to produce l‐asparaginase by improving gene expression and optimizing the fermentation agitation speed. Gene expression was improved by respectively increasing transcription levels and translation speeds through screening promoters and RBS sequences. With the optimal promoter, P43, and the synthetic RBS sequence, the yield obtained in a shake flask was 371.87 U/mL, which was 2.09 times that with the original strain. To further enhance production in a 5‐L fermenter, a multistage agitation speed control strategy was adopted, involving agitation at 600 rpm for the first 12 h, followed by a gradual increase in speed to 900 rpm, which resulted in the highest yield of l‐asparaginase, 5321 U/mL, after 42 h of fermentation. [ABSTRACT FROM AUTHOR]

Protein glycosylation is the most complex post‐translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery. Indeed, the carbohydrate residues in natural proteins affect their folding, immunogenicity, and stability toward proteases, besides controlling biological properties and activities. It is therefore important to introduce such structural modification in bioengineered proteins lacking the presence of carbohydrate residues. This is not trivial as it requires reagents and conditions compatible with the protein's stability and reactivity. This work reports on the introduction of lactose moieties in two natural proteins, namely ubiquitin (Ub) and l‐asparaginase II (ANSII). The synthetic route employed is based on the sulfur(VI) fluoride exchange (SuFEx) coupling of a lactose tethered arylfluorosulfate (Lact‐Ar‐OSO2F) with the ϵ‐NH2 group of lysine residues of the proteins. This metal‐free click SuFEx reaction relies on the properties of the fluorosulfate employed, which is easily prepared in multigram scale from available precursors and reacts chemoselectively with the ϵ‐NH2 group of lysine residues under mild conditions. Thus, iterative couplings of Lact‐Ar‐OSO2F to Ub and ANSII, afforded multiple glycosylations of these proteins so that up to three and four Lact‐Ar‐OSO2 groups were introduced in Ub and ANSII, respectively, via the formation of a sulfamoyl (OSO2‐NH) linkage. The metal‐free click SuFEx reaction was employed for the coupling of a lactosyl fluorosulfate with the free ϵ‐amino groups of lysine moieties incorporated into ubiquitin (Ub) and into the validated drug asparaginase II (ANSII). The glycosylation of ANSII, slowing down the clearance of the enzyme in vivo, increases its half‐life with a beneficial reduction of its administration frequency. [ABSTRACT FROM AUTHOR]

The article presents a case study related to 14-year-old boy presented with lymphadenopathy, petechiae, and hepatosplenomegaly. Topics include the patient has investigated for diabetes mellitus again because his glycemic control has worsened, the hyperglycemia often occurs during treatment of acute lymphoblastic leukemia (ALL) and has recognized the adverse effect of corticosteroids plus L-asparaginase (L-Asp), and the treatment of ALL, and dose intensification of L-Asp improves outcomes.

Abstract: The objective of the study was to achieve enhanced production of l‐asparaginase (LA), an anticancer enzyme, by a marine‐derived Aspergillus niger isolate. To improve LA production, optimization of pH, incubation time, and inoculum size was performed using process‐centric (response surface methodology [RSM]) and data‐centric (artificial neural network [ANN]) approaches. The optimized conditions led to a 108.62% rise in LA production. Upon comparison of 2 models for enhanced LA production, based on the R2, mean absolute percentage error, root mean square error, and mean absolute deviation values, the ANN model was observed to be superior over the RSM model. To the authors' best knowledge, the present finding is the first ever report revealing detailed analyses for RSM and ANN models for LA production using a marine‐derived fungus. [ABSTRACT FROM AUTHOR]

Recombinant proteins are revolutionizing present day therapeutics. They are generally expressed as insoluble inclusion bodies in the E. coli and mis‐folding, loss of protein, and high cost of down streaming are the hurdles in their recovery. For the first time, we are reporting the refolding with simultaneous purification of rhASP in E. coli using a single step utilizing protein folding‐strong anion exchange chromatography (PF‐SAX). The purification method is also standardized for optimal concentration of solution additives, pH, and mobile phase composition. The results showed purification of rhASP with anion exchange chromatography was effective. Phosphate buffer and slightly alkaline pH produced significant recovery yields and purity profiles. The effect of solution additives such as arginine, glycerol, TMAO, sorbitol, dextran, glutamate, and fructose on rhASP renaturation is also investigated. Significant results were achieved using arginine‐TMAO combination in terms of purity, recovery yield and specific activity of 99%, 78%, and 210 IU/mg, respectively. The work concludes that PF‐SAX refolding method is superior to other conventional methods and it can be applied to large scale purification of rhASP produced in E. coli. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1036–1044, 2018 [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: L‐asparaginase (ASNase) is an important biopharmaceutical used to treat the acute lymphoblastic leukemia (ALL) and lymphosarcoma. Considering its main use in cancer therapy, the most important request for ASNase production is the need for a highly pure biopharmaceutical obtained in the final of the downstream process, which is considered as the crucial step in its production. RESULTS: This work proposes the use of polymer–salt aqueous two‐phase systems (ATPS) based on polyethylene glycol and citrate buffer, with ionic liquids (ILs) as adjuvants, combined with the permeabilization of cell membrane using n‐dodecane and glycine for the in situ purification of periplasmatic ASNase from Escherichia coli cells. The process proposed was optimized (polymer molecular weight, pH, tie‐line length/mixture point, presence, nature and concentration of the adjuvant). The results show that ASNase partitions mostly to the PEG‐rich phase, due to hydrophobic interactions between both PEG and enzyme. Remarkably, the addition of 5 wt% of 1‐butyl‐3‐methylimidazolium methanesulfonate [C4mim][CH3SO3] as adjuvant lead to high recoveries [87.94 ± 0.03 (%)], purification factors (20.09 ± 0.35), and a final specific activity SA = 3.61 ± 0.38 U mg‐1 protein, from a crude enzyme extract with a SA = 0.18 ± 0.05 U mg‐1 protein. Moreover, better results were achieved when a prepurification step consisting of an ammonium sulfate precipitation was combined with the optimized ATPS, achieving an increased SA = 22.01 ± 1.36 U mg‐1 protein and PF = 173.8. CONCLUSIONS: A novel integrated downstream process was successfully implemented for the in situ purification of ASNase from fermentation broth. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract: The purification of recombinant L‐asparaginase II from an Escherichia coli fermentation broth was carried out by using solvent‐freeze‐out technology on the lab scale. Preliminary crystallization conditions were determined by using batch crystallization screening. The purification of L‐asparaginase II was performed afterwards. Protein crystals with rhombic shape were obtained from the solvent‐freeze‐out crystallization using ethanol. The purity of the crystals was evaluated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) together with UV spectrophotometry. It was found that DNA could be mostly removed by using solvent‐freeze‐out crystallization. Furthermore, the enzymatic activity was preserved. [ABSTRACT FROM AUTHOR]

Key Clinical Message: Myeloid/natural killer cell precursor acute leukemia (MNKPL) is a rare leukemia subtype characterized by a high incidence of extramedullary infiltration. No appropriate treatment strategy has so far been developed. Acute myelogenous leukemia‐type chemotherapy combined with L‐Asparaginase is an effective treatment for MNKPL. Hematopoietic cell transplantation is a second option in refractory cases. [ABSTRACT FROM AUTHOR]

l‐asparaginases ( EC 3.5.1.1) play an important role in nitrogen mobilization in plants. Here, we investigated the biochemical and biophysical properties of potassium‐dependent (PvAspG1) and potassium‐independent (PvAspG‐T2) l‐asparaginases from Phaseolus vulgaris. Our previous studies revealed that PvAspG1 requires potassium for catalytic activation and its crystal structure suggested that Ser‐118 in the activation loop plays a critical role in coordinating the metal cation. This amino acid residue is replaced by isoleucine in PvAspG‐T2. Reciprocal mutants of the enzymes were produced and the effect of the amino acid substitution on the kinetic parameters, allosteric effector binding, secondary structure conformation, and pH profile were studied. Introduction of the serine residue conferred potassium activation in PvAspG‐T2. Conversely, the PvAspG1‐S118I mutant could no longer be activated by potassium. PvAspG1 and the PvAspG‐T2‐I117S mutant had a similar half‐maximal effective concentration (EC50) value for potassium activation, between 0.1 and 0.3 m m. Potassium binding elicited a similar conformational change in PvAspG1 and PvAspG‐T2‐I117S, as studied by circular dichroism. However, no change in conformation was observed for PvAspG‐T2 and PvAspG1‐S118I. Analysis of kinetic parameters in function of pH indicated that potassium activation mediated by Ser‐118 influences the ionization of specific functional groups in the enzyme–substrate complex. Together, the results indicate that Ser‐118 of PvAspG1 is essential and sufficient for potassium activation in plant l‐asparaginases. Enzyme: l‐Asparaginase ( EC 3.5.1.1). [ABSTRACT FROM AUTHOR]

Abstract: Objectives: To establish the occurrence of increased plasma ammonia concentration after L‐asparaginase (L‐asp) administration in dogs with high‐grade lymphoma or leukemia; to identify risk factors for the development of hyperammonemia after L‐asp administration; and to determine occurrence of adverse events related to hyperammonemia. Design: Prospective case controlled study of sequentially enrolled dogs between May 2011 and March 2012. Setting: A university veterinary teaching hospital. Animals: Twenty‐seven dogs with high‐grade lymphoma or leukemia. Interventions: All dogs received L‐asp intramuscularly at a median dose of 400 IU/kg. Measurements and Main Results: Plasma ammonia concentrations were measured at baseline, 16 hours, and 48 hours after L‐asp therapy. Clinicopathological abnormalities were assessed to determine risk factors for the development of hyperammonemia. Adverse events following L‐asp were recorded. Median plasma ammonia concentrations at baseline, 16 hours, and 48 hours were 26 μmol/L (44 μg/dL), 98 μmol/L (166.9 μg/dL), and 67 μmol/L (114 μg/dL), respectively. Median plasma ammonia concentrations at 16 and 48 hours after administration were significantly increased compared to baseline. Six dogs had adverse events following L‐asp administration. No significant clinical signs were noted that could clearly be attributed to hyperammonemia. No risk factors for developing hyperammonemia were identified; however, there was a positive correlation between the development of hyperammonemia at 16‐ and 48‐hour time points. Conclusions: Subclinical hyperammonemia in dogs with lymphoma or leukemia after L‐asp administration appears to be common. No risk factors were identified for the development of hyperammonemia after L‐asp treatment, and severe adverse events were rare. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Nasal‐type extranodal natural killer NK/T‐cell lymphoma (ENKTCL) is a distinct type of non‐Hodgkin lymphoma with poor prognosis. This research aimed to evaluate the efficacy and safety of the GELOXD or P‐GEMOXD regimens in patients with ENKTCL. Methods: Newly diagnosed ENKTCL patients treated with either the GELOXD or the P‐GEMOXD regimen were identified from three cancer centers between January 2010 and December 2016. Kaplan‐Meier and Cox regression analyses were used to calculate overall survival (OS) and progression‐free survival (PFS) and to investigate prognostic factors. Results: One hundred and eighty‐four cases were identified from three cancer centers. After 1‐5 treatment cycles of GELOXD or P‐GEMOXD chemotherapy, 155 (84%) patients showed a complete response (CR). The 3‐year OS (73.0% vs 38.2%, P = .001) and PFS (72.8% vs 32.4%, P = .000) rates were significantly higher in early‐stage patients compared with advanced‐stage patients. A multivariate analysis revealed that patient CR status was a significant independent factor in disease prognosis. Grade 3/4 leukopenia occurred in 43 (23.4%) patients. Major non‐hematological toxicities included nausea (n = 117, 63.6%) and vomiting (n = 66, 35.9%). Conclusions: The GELOXD and P‐GEMOXD chemotherapy regimens are well tolerated and provide favorable survival outcomes in patients with ENKTCL. [ABSTRACT FROM AUTHOR]

Summary: Acrylamide in foods is declared as carcinogen. In the present work, the effect of enzymatic pretreatment and other parameters like enzyme concentration, frying conditions with respect to temperature and time, size of potato chips, and effect of sodium chloride and citric acid on mitigation of acrylamide were studied. The concentration of acrylamide in fried potatoes after the pretreatment was found to be 815.63 μg kg−1. The optimised asparaginase concentration for the mitigation of acrylamide was calibrated as 4 U mL−1, and optimised frying time and temperature were 15 min and 170 °C, respectively. An in‐depth kinetic relationship for the effect of asparaginase on the mitigation of acrylamide was studied. The prime novelty of the project is focused on the immobilisation of asparaginase to nanomagnetic particles for redundant usage with stabilised enzyme activity. The work projected three stables cycles of asparaginase activity and on further usage of the immobilised enzyme resulted in decreased activity. The repeated use of immobilised asparaginase provides the advantage of decreasing cost in processing. [ABSTRACT FROM AUTHOR]