A general approach for stabilizing nanobodies for intracellular expression

Area of Science:

• Biotechnology and Molecular Biology
• Immunology and Protein Engineering

Background:

• Conventional antibodies are too large for effective intracellular targeting in live cells.
• Nanobodies offer a smaller alternative for intracellular applications, but their stability is not well understood.
• A systematic study on the intracellular expression and stability of nanobodies is lacking.

Purpose of the Study:

• To systematically assess the intracellular expression and stability of a diverse set of nanobodies.
• To develop a generalizable method for enhancing the intracellular stability of nanobodies.
• To identify sequence features influencing nanobody stability within cells.

Main Methods:

• Screened 75 nanobodies from the Protein Data Bank for intracellular expression and stability.
• Employed comparative analysis and framework mutagenesis to engineer stabilized nanobodies.
• Validated stabilized nanobody expression in cellular and in vivo models (murine retina, E. coli).

Main Results:

• A majority of tested nanobodies exhibited instability, aggregation, and clearance when expressed intracellularly.
• Developed a generalizable mutagenesis approach that significantly improved the intracellular stability of most nanobodies.
• Identified specific sequence features critical for nanobody intracellular stability without compromising target binding.

Conclusions:

• Intracellular nanobody expression is often limited by inherent instability.
• A broadly applicable engineering strategy can overcome nanobody instability for intracellular use.
• Enhanced nanobody engineering will advance intracellular research and therapeutic interventions.