New strategy for the design and cloning of novel humanized biparatopic anti-CD52 nanobodies: Tools for targeted drug delivery and therapeutic approaches

Immunotherapy has been developed to enhance the effectiveness of targeted cancer treatments. CD52 as the appropriate cell surface marker is a glycoprotein anchored to the cell membrane. It is found in large quantities in cancerous lymphoid cells, particularly in B and T cells, making it a suitable target for such therapies. Anti-CD52 antibodies can induce cell lysis through complement activation and direct cell-mediated cytotoxicity. However, the immunogenicity of antibodies limits their application in human disease. In contrast, the distinct properties of nanobodies make them ideal tools for targeted therapy. Here, we developed a novel humanized biparatopic nanobody targeting CD52, referred to as huCD52. We grafted the amino acid sequences of the CDRs from Alemtuzumab (1ce1) and Gatralimab (6obd) into a general humanized nanobody scaffold (Caplacizumab). We also conducted molecular docking analysis and molecular dynamics simulations. The nanobody gene fragment was cloned into the pET-21b(+) vector, expressed in E. coli, and subsequently purified. We evaluated the binding activity of the nanobody using the ELISA test. Human biparatopic huCD52 nanobody showed a strong binding affinity to CD52 protein. Our approach is a versatile alternative to traditional protocols, which usually require immobilization or complex selection methods. We have developed a platform for constructing high-affinity, biparatopic nanobody-based constructs, which has the potential to accelerate the development of therapeutic interventions targeting CD52 in antigen-presenting leukemia.