(21c) Discovery and Characterization of Intracellularly Stable hnRNPA2B1 Specific Nanobodies for Live-Cell Imaging and Targeted Protein Degradation

Heterogeneous nuclear ribonuclear proteins, hnRNPs, play a variety of roles in regulating transcriptional, and post-transcriptional gene expression, including RNA splicing, polyadenylation, localization, translation, and turnover. A core member of the hnRNP family, hnRNPA2/B1, is a ubiquitously expressed nuclear protein known to shuttle between the nucleus and cytosol. Moreover, hnRNPA2/B1 proteins are also involved in the formation of membraneless-organelles such as stress granules (SGs) under certain conditions.

Mutations in hnRNPA2/B1 proteins are known to cause multisystem proteinopathy (MSP), a pleiotropic group of inherited disorders that cause neurodegeneration, myopathy, and bone disease. Mutations in hnRNPA2/B1 are also risk factors for the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Additionally, hnRNPA2B1 proteins are upregulated in numerous cancers including breast cancer, prostate cancer, and lung cancer. Despite growing evidence implicating hnRNPA2/B1 proteins in a multitude of diseases, our ability to study them in live cells is severely limited due to the lack of intracellularly stable hnRNPA2/B1 specific antibodies.

Nanobodies (sdAbs) are antibody fragments derived from heavy-chain-only antibodies found in camelids and sharks. Owing to their small size (~15 kDa) and unique biochemical properties, nanobodies are emerged as promising alternatives to conventional-antibody derived protein tools, such as single chain-variable fragments (scFvs). Their compact shape and convex antigen binding site (paratope), make nanobodies attractive alternative to probe surfaces that are not accessible to conventional antibodies. Furthermore, nanobodies have superior stability that facilitate their intracellular expression. Full length antibodies and bulkier antibody derived fragments, such as scFvs, are not amenable to intracellular expression, in part due to the reducing cytoplasmic environment that prevents the formation of disulfide bonds needed for their correct folding. The ability to express nanobodies intracellularly opens the door for studying protein-protein interactions, protein aggregations, and protein localizations in live cells under the microscope.

We have developed a high-throughput nanobody discovery platform from synthetic yeast surface display libraries by using antigen coated magnetics beads. Using this platform, we have identified novel hnRNPA2/B1 specific nanobodies and characterized their intracellular stability by expressing them in HEK293 cells. Furthermore, we have developed generalizable and straightforward cellular assays to characterize nanobody specificity in live cells. In addition to using nanobodies for studying hnRNPA2B1 biology in live cells, we have also engineered a panel of modular E3 ubiquitin ligases targeting hnRNPA2/B1 for degradation.

In this presentation, factors affecting successful high-throughput antibody discovery using yeast-surface display will be discussed. We will also talk about the intracellular stability of nanobodies and generalizable methods to characterize nanobody specificity in live cells. Furthermore, we will introduce protein engineering approaches to enhance nanobody affinity. Finally, we will discuss ways of using nanobodies for targeted protein degradation.