A Novel Anti-CD22 Camelid Immunotoxin Fused to Diphtheria Toxin for TargetedB-Cell Malignancy Therapy

Introduction: Hematologic malignancies, particularly of B-cell origin, represent approximately 6.5% of all cancers. While conventional therapies remain first-line treatments, targeted therapies such as immunotoxins (ITs) offer promising alternatives, especially in relapsed or refractory cases. ITs are fusion proteins combining a targeting ligand with a potent toxin. CD22, a B-cell surface marker with restricted expression and rapid internalization, is an attractive target for IT development. Methods: We developed a recombinant immunotoxin (rIT) by fusing a camelid-derived anti-CD22 nanobody (Nb22, ~15 kDa, ~400 bp) to a truncated diphtheria toxin (DT386, ~43 kDa, ~1,158 bp) via a human IgA1 hinge linker (~32 amino acids). The resulting fusion protein (~62 kDa, ~1,700 bp) was expressed in E. coli WK6 and characterized by SDS-PAGE and Western blot. Binding affinity to CD22 was evaluated by ELISA and flow cytometry. Cytotoxicity was assessed using MTT assays on Raji and Daudi (CD22⁺) and MOLT-4 (CD22⁻) cells. Results: The rIT exhibited high binding affinity to CD22 (Kaff ≈ 9.4 nM). Cytotoxicity assays demonstrated selective killing of Raji cells at low concentrations (1.6 nM), while MOLT-4 cells remained unaffected. In contrast, DT alone displayed non-specific toxicity only at high doses. Discussion: The selective cytotoxicity of the rIT demonstrates the potential of nanobody-based immunotoxins to target CD22⁺ cells while sparing non-target cells, highlighting an advantage over conventional toxin approaches. These results support further in vivo evaluation and optimization of Nb22-DT386 as a targeted therapy for B-cell hematologic malignancies. Conclusion: The developed rIT is a promising candidate for targeted therapy of CD22⁺ hematologic malignancies, offering high specificity, potent cytotoxicity, and potential for reduced off-target effects.