Design of a Soluble de novo CD20 Antigen for Therapeutic Antibody Discovery (2023)

Undergraduate: Kiara Thompson

Faculty Advisor: Brian Kuhlman
Department: Biochemistry & Biophysics

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Since 1997, Rituximab and other CD20-antibodies have been developed as monoclonal antibodies for treating illnesses like leukemia and non-Hodgkin lymphoma. CD20 is a transmembrane cellular protein expressed during B lymphocyte development and has remained an efficient target for B cell-generated malignancies. However, due to the insolubility of this protein, the native CD20 binding structure with therapeutic antibodies is challenging to study. These CD20 properties make antibody development methods, such as cell-surface display, inapplicable. In order to facilitate antibody drug development for leukemia, we used Rosetta, a computational protein design tool, to engineer a de novo antigen. This antigen maintains the native antigen binding features with altered features increasing the stability and solubility making it compatible for use in antibody development.

In order to validate the binding and stability of the de novo antigen, we expressed the de novo protein and anti-CD20 fragment antigen-binding (Fab) antibody including Rituximab, Obinutuzumab, and Ofatumumab. We used Biolayer Interferometry (BLI) to prove the affinity between the de novo antigen with Fabs. We also utilized Nano Differential Scanning Fluorimetry (nanoDSF) to demonstrate the stability of the de novo antigen. After validating the expression and binding of the de novo antigen with anti-CD20 Fragment antigen-binding antibodies (Fabs), the focus shifted to the expression of our de novo antigen in bacterial cells to gain a further understanding of its binding mechanism.

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