Isolation and characterization of human anti-CD20 single-chain variable fragment (scFv) from a Naive human scFv library

Abstract

CD20 is a receptor expressed on B cells with anonymous functions. The receptor is the target of some food and drug administration (FDA) approved monoclonal antibodies (mAb), such as Rituximab and Obinutuzumab. Blocking CD20 using the aforementioned mAbs has improved Non-Hodgkin Lymphoma (NHL) therapy. All commercial mAbs on the market were raised in non-human animal models. Antibody humanization is inevitable to mitigate immune response. In order to keep the affinity of antibody intact, humanizations are only applied to frameworks which do not eliminate immune response to foreign CDRs sequences. To address this issue, human monoclonal antibody deemed imperative. Herein, we report the isolation and characterization of a fully human single-chain variable fragment (scFv) against the large loop of CD20 from naïve human antibody library. After three rounds of phage display, a library of enriched anti-CD20 scFv was obtained. The polyclonal phage ELISA demonstrated that after each round of phage display, the population of anti-CD20 scFv became dominant. The scFv, G7, with the most robust interaction with CD20 was selected for further characterization. The specificity of G7 scFv was evaluated by ELISA, western blot, and flow cytometry. Detecting CD20 in western blot showed that G7 binds to a linear epitope on CD20 large loop. Next, G7 scFv was also bound to Raji cell (CD20+) while no interaction was recorded with K562 cell line (CD20—). This data attested that the epitope recognized by G7 scFv is accessible on the cell membrane. The affinity of G7 scFv was estimated to be 63.41 ± 3.9 nM. Next, the sensitivity was evaluated to be 2 ng/ml. Finally, G7 scFv tertiary structure was modeled using Graylab software. The 3D structure illustrated two domains of variable heavy (VH) and variable light (VL) connected through a linker. Afterward, G7 scFv and CD20 were applied to in-silico docking using ClusPro to illustrate the interaction of G7 with the large loop of CD20. As the selected scFv from the human antibody library is devoid of interspecies immunogenic amino acids sequences, no humanization or any other modifications are required prior to clinical applications.