scholarly journals Pre-Clinical Evaluation of Novel L-Asparaginase Mutants for the Treatment of Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4919-4919
Author(s):  
Soumika Sengupta ◽  
Mainak Biswas ◽  
Khushboo Gandhi ◽  
Vikram Gota ◽  
Avinash Sonawane

Abstract Introduction Acute Lymphoblastic Leukemia (ALL) accounts for 20% of all hematological malignancies. L-Asparaginase has been a mainstay of ALL for the last 6 decades and is also included in the WHO list of essential medicines for ALL. Escherichia coli L-asparaginase (EcA) was the first asparaginase to be approved for clinical use. However being isolated from bacteria, EcA has many side-effects which in turn affects the tolerability and efficacy of the drug. EcA administration may cause strong immunogenic and hypersensitive reactions in the patients, necessitating withdrawal of the drug. Sensitive individuals react to repeated EcA administration with formation of anti-drug antibodies (ADAs) that bind to and inactivate the enzyme leading to inadequate plasma levels of EcA. Another serious drawback of EcA is the glutaminase activity which leads to neurotoxicity. Other side effects include hepatotoxicity, thromboembolism and pancreatitis. Although a number of attempts have been made to alleviate these problems by rational protein engineering, the optimization of therapy with EcA for ALL patients still remains a challenge. In an attempt to deal with these problems, we created several EcA mutants. On the basis of their activity, stability and antigenicity we short-listed four EcA mutants (Mutant A, B, C and D) having favourable properties for further development. Methods We identified and mutated several B-cell epitopes and amino acid residues at the EcA interface that are responsible for activity, stability and antigenicity. Enzyme activity was measured at 37 oC (optimum temperature for EcA). Glutaminase activity of the mutants was measured and compared to the wild type EcA. The cytotoxicity of the EcA variants was verified in ALL sensitive REH cell lines by performing MTT assay after 24 h incubation. Further the antigenicity of the mutants was assessed by performing indirect ELISA where the binding of the mutants to the commercially available l-asparaginase antibody was analysed. Further, in vivo immunogenicity was evaluated by immunizing Balc C mice with primary and booster doses of EcA mutants over 66 days followed by the measurement of IgG and IgM titers. In addition, the binding of wild-type EcA and mutants to pre-existing anti-asparaginase antibodies in serum isolated from primary and relapsed ALL patients receiving asparaginase therapy was studied by indirect ELISA. Pharmacokinetics of the mutants was evaluated in female Balb C mice by plotting the asparaginase activity-time curve till 24 h following administration of a single i.v. dose of 50 IU/kg and compared with the wildtype. Finally the safety of the EcA mutants was determined by performing single-dose acute toxicity study at 3 dose levels in Balb C mice. Results At 37 oC, we did not find any significant difference in asparaginase activity of any EcA variant with the wild-type. All four variants showed markedly reduced glutaminase activity as compared to wild-type EcA (P<0.05). In MTT assay Mutant D showed 34.02%, Mutant B (32.4%), Mutant C (31.4%), Mutant A (24.22%), and wild type EcA (24.37%) reduction in REH cell viability in comparison to untreated cells. Binding to commercially available anti-asparaginase antibody was 49.09%, 32.63%, 27.43% less for Mutant D, Mutant B and Mutant C respectively compared to wild type EcA. Mice immunized with Mutant D showed 5-fold lower titres of IgG and 4-fold lower titres of IgM in comparison to wild type. Similarly, when compared to wild type, mice immunized with Mutant C showed 2.5-fold lower titres of IgG and 3.5-fold lower titres of IgM. At the same time Mutants B, C and D showed 2-3 fold less binding to pre-existing anti-asparaginase antibodies in samples collected from primary ALL patients undergoing asparaginase therapy. Similarly mutants B, C and D showed approximately 2-fold less binding to pre-existing anti-asparaginase antibodies in samples collected from relapsed ALL patients. Pharmacokinetic profiling showed that half life of Mutant A (267.28 ± 9.74), Mutant B (213.29 ± 6.53) and Mutant D (273.83 ± 35.45) was significantly longer than the wild type (102.17 ± 7.7). In acute toxicity study, we did not observe any significant toxicity of the mutants over the wildtype EcA. The findings are summarized in the figure. Conclusion Considering the immunogenicity, antigenicity and pharmacokinetics, mutant D emerged as a potent drug candidate for further development in the treatment of ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4912-4912
Author(s):  
Philip L Lorenzi ◽  
Wai Kin Chan ◽  
Andriy Anishkin ◽  
Preeti Purwaha ◽  
David M Rogers ◽  
...  

Abstract L-Asparaginase (L-ASP) is a key component of acute lymphoblastic leukemia therapy. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. In the present study, we tested the hypothesis that L-ASP glutaminase activity is required for anticancer activity. We first used molecular dynamics simulations of the clinically used E. coli L-ASP enzyme to guide engineering of mutants that lack glutaminase activity. Dynamic mapping of enzyme-substrate contacts identified the backbone amine of residue Q59 as having frequent contact with glutamine but not asparagine substrate. That difference identified Q59 as a promising mutagenesis target for modifying substrate selectivity. Saturation mutagenesis and screening of the resulting Q59 mutants identified Q59L as retaining asparaginase activity yet exhibiting undetectable glutaminase activity. Using Q59L to test the glutaminase-anticancer hypothesis, we observed no anticancer activity by Q59L against cell lines that do express asparagine synthetase (ASNS), including six leukemia lines—CCRF-CEM, SR, MOLT-4, K562, NALM-6, and REH—and two ovarian cancer lines—OVCAR-8 and SK-OV-3. Wild-type (WT) L-ASP, on the other hand, effected a dose-response in all of those cell lines, suggesting that glutaminase activity is required to kill cancer cells that express ASNS.  Unexpectedly, Q59L exhibited potent anticancer activity against cell lines that do not express detectable ASNS, including the leukemia cell lines Sup-B15 and RS4;11 and ASNS siRNA-treated OVCAR-8 cells. We conclude that the glutaminase activity of L-ASP is not necessary for anticancer activity against cell types that do not express ASNS. Since Q59L is expected to exhibit reduced toxicity relative to wild-type L-ASP because of its reduced glutaminase activity, these findings provide rationale for clinical assessment of Q59L L-ASP for the treatment of ASNS-deficient cancers. Disclosures: Lorenzi: ERYtech Pharma: Consultancy, Membership on an entity’s Board of Directors or advisory committees, US 7985548, US 7985548 Patents & Royalties. Off Label Use: L-asparaginase is an enzyme-drug approved for treatment of acute lymphoblastic leukemia.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1240-1240
Author(s):  
Jimei Zhao ◽  
Masahiro Ueki ◽  
Saori Sawai ◽  
Minako Sugiyama ◽  
Yukayo Terashita ◽  
...  

Abstract Background In the treatment of childhood acute lymphoblastic leukemia (ALL), 6-mercaptopurine (6-MP) is essential for early intensification and maintenance therapy. Recently, it has been reported that a gene polymorphism of nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) rs116855232 is associated with the 6-MP induced severe myelotoxicity. Since the NUDT15 rs116855232 polymorphism is relatively common in East Asian and Hispanic populations, it is important to evaluate the association between the polymorphism and 6-MP for determining the dose of effective therapy and avoiding the side effect in Japan. However, there are few reports on the association between NUDT15 polymorphisms and the therapeutic efficacy or side effects of 6-MP in the early intensification; most previous reports focused on the maintenance therapy so far. The purpose of this study is to clarify the association between NUDT15 polymorphism and the actual treatment status with 6-MP or its side effects, therefore to contribute to the effective and safe treatment of childhood ALL in Japan. Methods Twenty-four patients with ALL who received early intensification therapy according to the JPLSG ALL-B12 or JPLSG ALL-T11 protocol at the Department of Pediatrics, Hokkaido University Hospital, between April 2013 and May 2021 were included in the study. We retrospectively collected the clinical and laboratory data from the clinical records. And we also performed the sequence analysis of the exon 1 and 3 in NUDT15 gene Results Genetic analysis of NUDT15 showed no nucleotide changes other than rs116855232. Twenty patients were CC (wild-type), 4 patients were CT (heterozygous-variant), and 0 patients were TT (homozygous-variant). Of the 4 patients with heterozygous-variant, one patient received a reduced dose of 6-MP because of early onset myelotoxicity during early intensification therapy. In this patient, the total dose of 6-MP was 70% of the standard dose. All the 24 patients showed myelotoxicity and hepatotoxicity during early intensification therapy. Correlations between NUDT15 haplotype and side effects as myelotoxicity and hepatotoxicity were not significant. Discussion In the heterozygous-variant of NUDT15 rs116855232, the incidence and degree of myelotoxicity and hepatotoxicity during early intensification therapy did not differ from the wild-type. The international consortium (Clinical Pharmacogenetics Implementation Consortium Guideline, 2018) recommends that the dose of 6-MP should be reduced to 10mg/m2/day in patients with homozygous variant of NUDT15, however, the reduction of the starting dose of 6-MP in those with heterozygous variant is not recommended. Of note, these recommendations were made based on the data in the maintenance therapy. Our study suggests that the reduction of the starting dose may not be necessary in children with heterozygous variant. The NUDT15 rs116855232 polymorphism is common in Japan, with 10% of heterozygotes and 1.1% of homozygotes. In future study, a larger study on the association between NUDT15 polymorphism and treatment outcome including side effects in the early intensification is needed in East Asia to confirm the results of our study. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
pp. 107815522110171
Author(s):  
Rim Frikha ◽  
Moez Elloumi ◽  
Tarek Rebai ◽  
Hassen Kamoun

Introduction Functional variants of the Methylenetetrahydrofolate reductase ( MTHFR) gene, the C677T and A1298C, have largely investigated in pharmacogenomics of Methotrexate (MTX) in acute lymphoblastic leukemia (ALL), yet the conclusions are inconsistent. In addition; most of these studies do not analyze haplotypes. Here, we investigate the MTHFR 677/1298 genotypes and the 677-1298 haplotype and characterize the MTX response in Northern African ALL patients. Methods Genomic DNA was extracted from whole venous from a total of 28 patients with ALL. Genotyping were carried out with restriction fragment length polymorphism (RFLP). A toxicity score (TS) is calculated for each patient and correlate to the haplotype. Results The allelic frequency of MTHFR 677T-1298C haplotype was 10.7% in ALL patients. According to the toxicity’s score (TS) there was no significant differences between haplotype groups (p = 0.79): TS was higher with wild type of MTHFR (TS = 3.43; SEM ± 0.85) followed by combined genotype (677T-1298C) (TS = 2.67; SEM ± 0.88) and isolated variant (C677T or A1298C) (TS = 2.64; SEM ± 0.92). Conclusion Despite the limitation of this study; our results suggest that the MTHFR 677T-1298C haplotype is common in ALL and may be a promising HD-MTX chemotherapy-related adverse effects biomarker.


Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3417-3423 ◽  
Author(s):  
Marina Bousquet ◽  
Cyril Broccardo ◽  
Cathy Quelen ◽  
Fabienne Meggetto ◽  
Emilienne Kuhlein ◽  
...  

Abstract We report a novel t(7;9)(q11;p13) translocation in 2 patients with B-cell acute lymphoblastic leukemia (B-ALL). By fluorescent in situ hybridization and 3′ rapid amplification of cDNA ends, we showed that the paired box domain of PAX5 was fused with the elastin (ELN) gene. After cloning the full-length cDNA of the chimeric gene, confocal microscopy of transfected NIH3T3 cells and Burkitt lymphoma cells (DG75) demonstrated that PAX5-ELN was localized in the nucleus. Chromatin immunoprecipitation clearly indicated that PAX5-ELN retained the capability to bind CD19 and BLK promoter sequences. To analyze the functions of the chimeric protein, HeLa cells were cotransfected with a luc-CD19 construct, pcDNA3-PAX5, and with increasing amounts of pcDNA3-PAX5-ELN. Thus, in vitro, PAX5-ELN was able to block CD19 transcription. Furthermore, real-time quantitative polymerase chain reaction (RQ-PCR) experiments showed that PAX5-ELN was able to affect the transcription of endogenous PAX5 target genes. Since PAX5 is essential for B-cell differentiation, this translocation may account for the blockage of leukemic cells at the pre–B-cell stage. The mechanism involved in this process appears to be, at least in part, through a dominant-negative effect of PAX5-ELN on the wild-type PAX5 in a setting ofPAX5 haploinsufficiency.


2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Saleh A. Mohamed ◽  
Mohamed F. Elshal ◽  
Taha A. Kumosani ◽  
Alia M. Aldahlawi

L-asparaginase from bacteria has been used in treatment of acute lymphoblastic leukemia. The aim of this study was to purify and characterize L-asparaginase fromPhaseolus vulgarisseeds instead of microbial sources. L-asparaginase was purified to apparent homogeneity. The enzyme has molecular mass of 79 kDa. The purified asparaginase had very low activity toward a number of asparagine and glutamine analogues. L-asparaginase was free from glutaminase activity. Kinetic parameters, Km andVmax of purified enzyme, were found to be 6.72 mM and 0.16 μM, respectively. The enzyme had optimum pH at 8.0. The enzyme showed high stability at alkaline pH (pH 7.5–9.0) when incubated for up to 24 h. L-asparaginase had the same temperature optimum and thermal stability at 37°C. K+was able to greatly enhance the activity of asparaginase by 150% compared with other metals tested. In conclusion, L-asparaginase showed no glutaminase activity and good stability over a wide range of physiological conditions, and thus it could be used as a potential candidate for treatment of acute lymphoblastic leukemia.


2009 ◽  
Vol 28 (8) ◽  
pp. 822-828 ◽  
Author(s):  
Claudio Giuseppe Molteni ◽  
Giovanni Cazzaniga ◽  
Daniele F. Condorelli ◽  
Cosimo G. Fortuna ◽  
Andrea Biondi ◽  
...  

2004 ◽  
Vol 26 (4) ◽  
pp. 217-226 ◽  
Author(s):  
Eduard H. Panosyan ◽  
Nita L. Seibel ◽  
Sagrario Martin-Aragon ◽  
Paul S. Gaynon ◽  
Ioannis A. Avramis ◽  
...  

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