Synthesis of Immunotoxin of Anti-CD3 Monoclonal Antibody-Bongkrekic Acid and Cytotoxic Effect Against CD3+ T Cells in Peripheral Blood Mononuclear Cells

Document Type : Research Articles

Authors

1 Department of Nutrition, Faculty of Psychology and Health, Universitas Islam Negeri Walisongo, Semarang, Indonesia, Formerly Affiliated with the Master’s Programme in Biomedical Science, Faculty of Medicine, Universitas Indonesia, 10430, Indonesia.

2 Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, 10430, Indonesia.

Abstract

Introduction: Cancer is a major health concern worldwide. Common cancer treatments such as surgery, chemotherapy, and radiotherapy have not been able to completely reduce the rate of cancer-related death. Monoclonal antibodies (MAb) have been widely used for cancer treatment in the form of immunotherapy or targeted therapy. Immunotoxins are a form of targeted therapy based on monoclonal antibody-toxin conjugates. Antibodies deliver toxins to specific cancer cells and induce cell death. Objective: This study aimed to synthesize a conjugate of bongkrekic acid (BKA), a potent mitochondrial toxin, with anti-CD3 MAb and evaluate its specificity in peripheral blood mononuclear cells (PBMC).Methods: In silico assays were performed to predict conjugation sites and interactions between BKA and MAb. The synthesis of the conjugate was carried out chemically using EDC-HCl/Sulfo-NHS zero-length crosslinker and confirmed using a UV-Vis spectrophotometer. PBMC were used as a specificity test model. The conjugate exhibited selective cytotoxic toward CD3+ T cells without affecting other cells in PBMCs.Results: In silico assays using molecular docking showed conjugation sites in the cavity of the CH2-CH3 Fc IgG2a domain and covalent interactions with lysine, asparagine, and glutamine amino acids. Measurements of absorption at wavelengths of 280 and 260 nm indicated the presence of protein and BKA in the synthesized conjugate. Incubation of PBMC with BKA and anti-CD3 MAb resulted in a significantly lower average number of cells (p< 0.05) than that observed in the group treated with the conjugate.Conclusion: In silico assays revealed an interaction between the carboxylic groups of BKA and the primary amine group of antibodies. The Conjugates exhibited lower cytotoxicity compared to BKA and anti-CD3 Mab individually. In vitro assays have not been able to show the specificity of the conjugate due to the anti-CD3 MAb alone exhibiting cytotoxic properties in PBMCs.

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Asian Pacific Journal of Cancer Prevention
Volume 27, Issue 2
February 2026
Pages 443-451

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History

  • Receive Date: 05 December 2022
  • Revise Date: 15 July 2025
  • Accept Date: 26 January 2026

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