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A Novel Anti-CD22 scFv.Bim Fusion Protein Effectively Induces Apoptosis in Malignant B cells and Promotes Cytotoxicity

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Abstract

CD22 is a B-cell surface antigen which is highly expressed in cancerous B-cell lineages. Anti-CD22 antibodies are currently under focus as promising biologics against hematologic B-cell malignancies. Herein, we introduce a novel active recombinant anti-CD22 scFv.Bim fusion protein for targeting this cancerous antigen. An expression cassette encoding anti-CD22 scFv.Bim fusion protein was expressed in Pichia pastoris. The binding ability, cytotoxicity, and apoptotic activity of the purified recombinant protein against CD22+ Raji cell line were assessed by flow cytometry, microscopy, and MTT assay. Using bioinformatics, the 3D structure of the fusion protein and its interaction with CD22 were assessed. The in vitro binding analysis by immunofluorescence microscopy and flow cytometry demonstrated the specific binding of scFv.Bim to CD22+ Raji cells but not to CD22− Jurkat cells. MTT data and Annexin V/PI flow cytometry analysis confirmed the apoptotic activity of anti-CD22 scFv.Bim against Raji cells but not Jurkat cells. In silico analysis also revealed the satisfactory stereochemical quality of the 3D model and molecular interactions toward CD22. This novel recombinant anti-CD22 scFv.Bim fusion protein could successfully deliver the pro-apoptotic peptide, BIM, to the target cells and thus nominates it as a promising molecule in treating B-cell malignancies.

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All data generated or analyzed during this study are included in the article (and its supplementary information files).

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Acknowledgements

The authors wish to thank Prof. Marjan Mohammadi for her excellent scientific comments on this work.

Funding

This work has been a part of the Ph.D. project of Zahra Mohammadi and financially supported by the Pasteur Institute of Iran.

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Authors and Affiliations

  1. Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, 12th of Farvardin Jonoobi Ave, Jomhoori Street, Tehran, Iran

    Zahra Mohammadi, Somayeh Enayati, Najmeh Zarei,  Mozhgan Zahmatkesh, Samaneh Saberi, Mohammad Azizi & Vahid Khalaj

  2. Thalassemia & Hemoglobinopathy Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

    Ladan Mafakher

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Contributions

Z.M. performed the experiments and wrote the first draft of the manuscript. S.E. and M.Z. helped in yeast expression studies. N.Z. assisted in cell-based assays. S.S. contributed to statistical analysis. L.M. assisted in bioinformatics section. M.A. supervised the molecular cloning procedures. V.K. designed and supervised the whole study and critically revised and finalized the manuscript.

Corresponding authors

Correspondence to Mohammad Azizi or Vahid Khalaj.

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Mohammadi, Z., Enayati, S., Zarei, N. et al. A Novel Anti-CD22 scFv.Bim Fusion Protein Effectively Induces Apoptosis in Malignant B cells and Promotes Cytotoxicity. Appl Biochem Biotechnol 194, 5878–5906 (2022). https://doi.org/10.1007/s12010-022-04035-y

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  • DOI: https://doi.org/10.1007/s12010-022-04035-y

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