Single-chain variable fragment (scFv) is the smallest functional unit of monoclonal antibody with a non-native peptide loop (linker) holding variable domain together. Its small size allows bacterial expression making engineering easy, and further gains pharmacokinetic advantages such as better tumor penetration and reduced immunogenicity. However, despite the great therapeutic potential, scFvs have not made it into the clinic due to a decrease in stability and affinity. Nearly all scFvs have been engineered with flexible (Gly4Ser)n linkers, which are susceptible to proteolysis leading scFvs to poor stability. Yet there has been no comprehensive study on compositional effects of the linker. Furthermore, binding maps of each complementarity determining region (CDR) loop are unavailable, which are required for affinity engineering. Here, we explored the loop compositions to enhance biophysical properties of 3E8, a Tumor-Associated Glycoprotein (TAG)-72 specific scFv. (1) Linker. Sets of linker libraries in varying compositions were constructed and characterized to determine favorable linker trends in scFvs. Based on this, a much broader sequence space was explored via combinatorial linker library and phage display, a high-throughput screening method. After the fifth round of enrichment, the library was deep sequenced, and the hits appearing in repeat were further characterized to confirm stability enhancements. The results showed significant increase in expression level and stability with high frequency of conformationally constrained amino acids. (2) CDR loops. Six CDR loops were alanine scanned to map the binding hotspot of 3E8 not only to improve affinity against TAG-72 but also to alter specificity to different cancer-related glycans. Based on this, loop libraries randomized at key residues were constructed. Fluorescence anisotropy based binding assay was developed to perform high-throughput screening against commercially available O-glycans. Taken together, this study provides invaluable insight into scFv engineering that is currently lacking in order to expand its utility as cancer therapeutics.

Exploring composition of peptide loops to enhance biophysical properties of antibody fragments for cancer therapeutics

2019 Fall National Meeting

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