Expression and functional characterization of an anti-CD22 scFv targeting B-cell malignancies

Monireh Gholizadeh , Shahriyar Abdoli, Shafieeh Mansoori, Arash Arashkia, Farhad Riazi-Rad, Amir Ali Hamidieh, Mohammad Nouri , Zahra Sharifzadeh

Abstract


Background and purpose: Single-chain variable fragments (scFvs) offer advantages over full-length monoclonal antibodies in cancer therapy, including reduced size, lower production costs, and easier handling. However, Escherichia coli (E. coli) often leads to the formation and aggregation of inclusion bodies (IBs). This study aimed to optimize the expression and purification of an anti-CD22 scFv (CD22-scFv) in E. coli and evaluate its functional properties.

Experimental approach: The CD22-scFv construct was subcloned into pET-28a(+) and expressed in E. coli strains Rosetta (DE3) and Rosetta-gami 2. To overcome IBs formation, two purification methods were employed to enhance soluble protein production: hybrid conditions, a novel one-step immobilized metal affinity chromatography (IMAC)-based on-column refolding method was employed, using gradually decreasing urea and increasing imidazole concentrations; native conditions, expression parameters (IPTG concentration, post-induction temperature, and time) were optimized, followed by IMAC. The CD22-scFv binding to CD22 antigen and its anti-proliferative effects on target cells were assessed via flow cytometry and MTT assay.

Findings/Results: CD22-scFv was successfully expressed in Rosetta (DE3) but not Rosetta-gami 2. Hybrid purification yielded 15.86 mg/L protein, outperforming native purification (3.65 mg/L). Flow cytometry confirmed the binding of native- and hybrid-purified CD22-scFv to CD22 Raji cells with 75.5% and 55.8% efficiency, respectively. Native-purified CD22-scFv significantly inhibited Raji cell proliferation while sparing CD22− cells.

Conclusion and implications: This study established a scalable and cost-effective strategy for producing functional CD22-scFv with high specificity and anti-proliferative effects. The findings highlight its potential for targeted therapies and diagnostics, warranting further in vivo and clinical studies.

 

 


Keywords


CD22-scFv; E. coli expression system; IMAC purification; Immunotherapy; On-column refolding; Protein solubility.

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