Talk abstracts
Talk on Wednesday 09:15-09:30am submitted by Nicholas Long
Characterization and Selection of Biophysically Optimized Antibody Fragments for Enhanced in vivo Tumor Imaging
N. Emerson Long (Ohio State Biochemistry Program), Brandon J. Sullivan (Ohio State Biochemistry Program), Thomas J. Magliery (Ohio State Department of Chemistry and Biochemistry)
Abstract:
Antibody fragments have great potential for clinical application as cancer theranostics. Their small size compared to full-length IgG’s allows for faster blood clearance, potentially decreased immunoreactivity, better tumor penetrance, and easier engineering and production. The smallest possible fragment of an IgG that still binds to its antigen, called the single-chain variable fragment (scFv), can be created by fusing the variable light and variable heavy domains together with a peptide linker. Along with switching domain orientations, altering the length and amino acid sequence of the linker can significantly change the biophysical characteristics such as binding, stability, and quaternary structure. Comprehensive studies of these attributes have not been reported in the literature, making design and optimization of antibody fragments challenging. Here we constructed linker and orientation libraries of 3E8, an antibody specific to TAG 72, a mucinous glycoprotein overexpressed in 80% of adenocarcinomas.
Our studies have confirmed dramatic differences based on linker and orientation choices regarding biophysical properties and in vivo imaging. Specifically we have cloned, expressed, and characterized scFVs, diabodies, and higher order multimer constructs with varying linker compositions and sizes and domain orientations. These constructs were characterized by surface plasmon resonance (SPR) to test for antigen binding, by differential scanning fluorimetry (DSF) to test for thermal stability, and by gel chromatography to test for quaternary structure and homogeneity. We then optimized expression and purification of two biophysically favorable constructs, 3E8.scFV and 3E8.G4S. Both constructs were subjected to mouse biodistribution and pharmacokinetic studies. From this analysis, we selected 3E8.G4S as a lead candidate for cancer imaging and detection. This hypothesis was confirmed with successful PET and SPECT imaging of cancer xenograft mice at 24 hours.
Keywords: Antibody fragments, Cancer diagnostics, Protein engineering