Construction and affinity enhancement of a new anti-CD۲۰ single-chain variable antibody fragment

Backgrounds: The B-lymphocyte antigen CD۲۰ exists on the surface of normal and mostmalignant B-cells. CD۲۰ is responsible for the regulation of calcium influx, and is able to startintracellular signaling pathways. The technology of monoclonal antibody (mAb) led to aneffective targeted therapy for a variety of diseases like autoimmune diseases and cancer. Anti-CD۲۰ mAbs could be applied in the treatment of various diseases. Rituximab combined withchemotherapy has revolutionized the treatment of CD۲۰+ B-cell non-Hodgkin lymphomas, butpatients often relapse. Despite the recent advances in development of anti-CD۲۰ mAbs,challenges remain to develop novel antibodies with improved properties.Materials and Methods: We have already proposed DNA-based immunization for theproduction of pharmacologically active anti-CD۲۰ mAbs, with consequent development of D۴mAbs against the native extracellular domain of CD۲۰ with new desirable characteristics. Themajor aim of the present study is to isolate genes encoding D۴ variable domains to design asingle-chain variable antibody fragment (scFv), in turn, allowing the conduct of studies onintermolecular interactions between CD۲۰ epitopes and D۴. Through a computational designapproach, the sequence was first immune-annotated and used to construct a model of scFvstructure. An accurate model of CD۲۰ was then built and docked to the scFv. Extensivestructural analyses were performed to identify appropriate substitution positions, followed by acomprehensive free energy-based mutation screening to enhance the scFv-CD۲۰ interactionaffinity.Results: In epitope motif and binding orientation analyses, the antibody and the derivedfragment showed the character of type II antibodies. Affinity enhancement screening identifiedthe novel triple-mutant scFv variant, H: A۶۴F/H: E۱۰۸R/L: I۳۷R, representing a more favorableantibody-antigen binding.Conclusion: Altogether, the data reported here support future subsequent procedures to producelarge quantity of recombinant D۴ anti-CD۲۰ antibodies, which can be utilized in therapy anddiagnosis of malignancies.

کلیدواژه ها:

Affinity enhancement ، CD۲۰ ، Epitope ، In silico design ، Single-chain variable antibody fragment