Engineering Nanobody Intracellular Behavior Through Framework Mutagenesis

Abstract

Antibodies and their derived fragments have traditionally been used to probe, neutralize, or interrogate primarily extracellular targets in living systems. While efforts have been taken to adapt the antigen-binding portions of conventional antibodies to the intracellular environment, a lack of consistent stable and soluble behavior has hampered their utility for intracellular studies in live cells. Nanobodies, variable domains derived from a special class of antibody lacking light chains, hold promise as intracellularly functional tools. At less than one-tenth the size of a conventional antibody, nanobodies retain full antigen binding function, and exhibit superior expressibility in the reducing environment of the cytoplasm. Furthermore, previous work in the Cepko lab has illustrated that nanobodies can be engineered to rely on antigen interaction for stability, a property facilitating the design of conditionally active tools that only function in cells expressing specific targets. In this dissertation, the generality of both intracellular stability and engineered, conditional instability are examined. In chapter 2, I highlight intracellular stability differences observed across a broad repertoire of previously identified nanobodies, interrogate sequence differences between intracellularly stable and unstable nanobodies, and develop a general approach to mutationally stabilize previously unstable nanobodies for intracellular expression. In chapter 3, I propose specific framework mutations, identified by mutational screens over nanobodies exhibiting disparate modes of target interaction, that facilitate both destabilization in the absence of antigen, and re-stabilization in the presence of antigen, transmissible across a broad range of nanobodies. This work will streamline the design and function of target-specific, intracellularly active protein tools.