development and characterization of a novel spytagged modular nanobody as a detection platform for cd22-positive cells

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 cytometry.results: 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).