Development and Characterization of A Novel SpyTagged Modular Nanobody as A Detection Platform for CD22-Positive Cells

Abstract

Objective: CD22, as a surface protein of B cells, is used in the diagnosis and target-specific immunotherapy of B-cellmalignancies. SpyTag and SpyCatcher, on the other hand, are two covalently coupled proteins capable of developinga bi- or multi-specific modular protein. The aim of this study was to develop FITC-conjugated SpyCatcher-SpyTaggedanti-CD22 Nanobody (FITC-SpyC-SpyT-CD22Nb) to recognize CD22 on the surface of malignant B cells.Materials and Methods: In this experimental study, the SpyTag-CD22Nb construct was subcloned into a pET22 vectorand expressed in E. coli BL21 (DE3). After purification using His-tag affinity chromatography, the size of the elutedprotein was confirmed on a Western blot. In addition, the SpyCatcher protein, subcloned into pET28, was expressed inE. coli BL21 (DE3), purified by His-tag affinity chromatography and subjected to FITC labeling. FITC-SpyCatcher andSpyTag-CD22Nb were coupled in a 1:1 molar ratio. The specific binding of the produced FITC-SpyC-SpyT-CD22Nbwas tested using CD22+ Raji and CD22- K562 cell lines and was evaluated by flow cytometryResults: SpyTag-CD22Nb and SpyCatcher were successfully expressed in E. coli BL21 (DE3). The 1:1 molar ratio ofSpyTag-CD22Nb and FITC-SpyCatcher successfully formed FITC-SpyC-SpyT-CD22Nb at room temperature. The flowcytometry results showed that FITC-SpyC-SpyT-CD22Nb specifically binds to the CD22+ Raji cells, while there is nobinding to the CD22- K562 control cells.Conclusion: The novel FITC-SpyC-SpyT-CD22Nb produced in the present study is capable of detecting the surficialexpression of CD22. According to our findings, FITC-SpyC-SpyT-CD22Nb is applicable for specific targeting of CD22in a therapeutic manner, i.e., chimeric antigen receptor (CAR)-T cell therapy and antibody drug conjugates (ADCs).

Keywords

• cd22
• molecular imaging
• single-domain antibody
• spycatcher
• spytag