Development of a biparatopic-domain antibody screening system

The mature form of human mesothelin (MSLN) is a cell-surface protein that is normally expressed at a low level on the mesothelial cells but is overexpressed in many cancers, including lung adenocarcinoma and mesothelioma, making it a practical target for therapeutic antibody applications. Our long-term goal, to develop MSLN-targeting domain antibodies that will be utilized to design therapeutic reagents. MSLN is highly relevant to environmentally caused disease and human health. In this research, we propose to develop a screening system for biparatopic human heavy chain (VH) domains in the tandem VH1-VH1- form, to find a better MSLN binder and to test whether biparatopic screening will identify novel binders compared to tethered VH domains generated using individually screened VHs. In Aim 1, we will identify binding sites of the tethered VH1-VH2- that were generated using the individually screened VHs. In Aim 2, we will develop a direct screening system of the biparatopic antibody domain in the VH1-VH2- form. Results obtained in each Aim will be independently fruitful to develop MSLN antibodies. We will also compare VH1-VH2 sequences and MSLN-binding sites obtained in Aim 2 with those in Aim 1. By identifying how the sequences and binding site are changed by the screening in Aim 2, we will scale up anti-MSLN VH1-VH2 screening and their characterization, including optimization of biparatopic CAR-T cell design, immunoncology and animal studies, and [in the future] plan to generalize the biparatopic domain antibody screening approach to other important tumor-associated antigens.