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Engineered L-asparaginase variants with enhanced therapeutic properties to improve treatment of childhood acute lymphatic leukemia

Cancer Gene Therapy volume 32, pages 1062–1075 (2025)Cite this article

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Abstract

Escherichia coli L-asparaginase (EcA), a key component of a multi-drug acute lymphatic leukemia (ALL) treatment regimen, has several limitations that reduce its therapeutic efficacy. The major disadvantages include immunogenicity, serum instability, shorter half-life, and accompanying glutaminase activity that causes neurotoxicity and pancreatitis. Pegylated asparaginase and Erwinase have better therapeutic potential, but they are expensive. Using site-directed mutagenesis, we created several EcA variants by substituting specific amino acid residues at the dimer-dimer interface and B-cell epitope regions. After several rounds of screening and selection, we identified two EcA variants viz. K288S/Y176F (KSY-17) and K288S/Y176F/W66Y (KSYW-17), which showed comparable asparaginase activity to wild-type (WT) and significantly less glutaminase activity (30.36 U/mg for WT vs 1.54 and 0.99 U/mg for KSY-17 and KSYW-17). KSYW-17 was less immunogenic than WT, eliciting 4.8–5.3-fold and 2.4–3.8-fold less IgG and IgM responses, respectively. Compared to WT EcA, we also observed significantly less (~1.5-2-fold) binding of these variants to pre-existing antibodies in ALL patients’ serum. Pharmacokinetic studies showed that KSY-17 (213.3 ± 6.5 min) and KSYW-17 (244.8 ± 35.5 min) had longer plasma half-lives than WT (101.1 ± 5.1 min). Both variants showed no toxicity up to 5000 IU/kg (single dose) and 1600 IU/kg (repeat dose) in mice. ALL xenograft mice studies showed a 90% and 70% reduction in leukemia burden in KSY-17 and KSYW-17 administered mice, respectively, as compared to 30% for WT after repeat dose administration, accompanied by significantly higher mice survival (100% vs. 70% vs. 10% for KSY-17 vs. KSYW-17 vs. WT). Overall, the engineered EcA variants’ showed improved therapeutic efficacy, thus making them promising candidates for primary and relapsed ALL treatment.

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Fig. 1: In-vitro properties of double variant KSY-17 and its subsequent triple variant KSYW-17.
Fig. 2: EcA’s immunogenic properties are altered by mutations.
Fig. 3: The wild-type EcA binds to pre-existing antibodies circulating in ALL patients undergoing asparaginase treatment, but not the variants.
Fig. 4: EcA variants with better in-vivo pharmacokinetics are better therapeutic candidates.
Fig. 5: Cathepsin B (CTSB) degrades the KSYW-17 triple variant but not the KSY-17 double variant.
Fig. 6: EcA wild-type and variants treated mice liver tissue for histopathology using Haematoxylin and Eosin (H&E) staining.
Fig. 7: Efficacy of wild-type EcA and variants in cell line derived leukemic xenograft model.

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The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.

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Acknowledgements

We are thankful to all members of AS and VG laboratories for valuable discussions. We would like to acknowledge Prof. Chitra Mandal (CSIR-IICB, Kolkata) for helping us in performing glutaminase assay and enzyme kinetic experiments and Dr H J Sharath Kumar for helping us in constructing pharmacokinetic graphs. We would also like to acknowledge Saroj Prasad Panda (Pediatric Hemato Oncologist, Bhubaneswar) for providing us with ALL patient samples and Dr Anand Patil (Scientific Officer C, ACTREC) for helping us in calculating the Pharmacokinetic parameters using PUMAS software. In-vivo pre-clinical studies were carried out using the animal facility, cell culture, microscopy and FACS at ACTREC, TMC. Spectrophotometric analysis was done at IICB, Kolkata. No drugs apart from laboratory-generated asparaginase were used in this study.

Funding

This work was supported by DST-SERB Grants, Government of India (SB/SO/HS/203/2013, CRG/2019/001593 and TTR/2022/000024) to Avinash Sonawane. UGC-NET and ICMR-SRF fellowships awarded to MB and SS, respectively is highly acknowledged.

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  1. These authors contributed equally: Mainak Biswas, Soumika Sengupta.

Authors and Affiliations

  1. School of Biotechnology, Campus-11, KIIT Deemed to be University, Bhubaneswar, Odisha, India

    Mainak Biswas, Soumika Sengupta, Manaswini Jagadeb & Avinash Sonawane

  2. Department of Clinical Pharmacology, ACTREC, Tata Memorial Centre, Navi Mumbai, India

    Khushboo A. Gandhi, Saurabh Kumar Gupta & Vikram Gota

  3. Homi Bhabha National Institute, Training School Complex, Mumbai, India

    Saurabh Kumar Gupta, Poonam B. Gera & Vikram Gota

  4. Department of Pathology, ACTREC, Tata Memorial Centre, Navi Mumbai, India

    Poonam B. Gera

  5. Department of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore (IIT Indore), Simrol, Madhya Pradesh, India

    Bhagyashri Soumya Nayak & Avinash Sonawane

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  1. Mainak Biswas
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Contributions

AS, MB, and SS conceived and administrated the project. MB and SS contributed equally to this study. MB and SS planned the experimental setup, performed the experiments, analyzed the data, and wrote the manuscript. BN helped in writing the manuscript. KG and VG planned pharmacokinetic studies and analyzed the data. SKG helped SS and KG in pre-clinical efficacy and toxicity study in mouse models. PG analyzed all the histology slides. MJ created the 3D image. AS and VG supervised the project, planned the experimental setup, analyzed the data, wrote the manuscript, and provided all the necessary resources and support for the completion of the study. All authors reviewed the results and approved the final version of the manuscript.

Corresponding authors

Correspondence to Vikram Gota or Avinash Sonawane.

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Ethical approval and consent to participate

Animal experiments were performed in accordance with the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines and approval of the Institutional Animal Ethical Committee (IAEC) was obtained. Institutional Ethical Committee (IEC) approval from Acharya Harihar Regional Cancer Center (AHRCC), Cuttack, Odisha, India (Approval Letter no: 067-IEC-AHRCC) was acquired and written informed consent was obtained from the study participants before taking the samples.

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Biswas, M., Sengupta, S., Gandhi, K.A. et al. Engineered L-asparaginase variants with enhanced therapeutic properties to improve treatment of childhood acute lymphatic leukemia. Cancer Gene Ther 32, 1062–1075 (2025). https://doi.org/10.1038/s41417-024-00865-6

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