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Engineered Nanobodies That Can Enter Cells to Unlock New Drug Targets

At a Glance

Researchers at Colorado State University have developed resurfaced cell-penetrating nanobodies that can enter living cells efficiently. These engineered proteins act as flexible scaffolds for discovering therapies that target disease-related molecules inside cells. Unlike most protein therapeutics, these nanobodies can reach intracellular receptors that were previously inaccessible. This platform increases the number of biological targets available for drug discovery and research.

Background

Many diseases are driven by proteins inside cells, but most protein-based drugs cannot cross the cell membrane. As a result, current biologics interact with proteins on the cell surface, which limits treatment options. Nanobodies are small, stable proteins that can bind targets with high specificity and are easily modified. Making them able to enter cells opens new possibilities for developing therapies and studying intracellular processes.

Overview

This platform uses resurfaced nanobodies engineered with positive surface charges to help them pass through the cell membrane. Nanobodies are small, robust antibody-derived proteins with strong binding ability. By modifying the surface while keeping their shape and stability, researchers created nanobodies that can enter cells and reach the cytosol without losing function.

These nanobodies can be used as general scaffolds. They can be screened or modified to bind different intracellular disease targets. Their stability and adaptability let researchers apply standard protein engineering techniques to discover new therapeutics. This platform allows drug discovery for targets that were previously hard to reach and support both lab-based (in vitro) and living organism (in vivo) testing.

Flow cytometry histograms comparing cellular uptake of GFP-tagged nanobodies. Engineered nanobodies (pcNB1-GFP; red, green, and blue curves) show progressively higher intracellular fluorescence as concentration increases, indicating dose-dependent cell entry. In contrast, unmodified NB1-GFP (gray) and GFP alone (black) display minimal fluorescence, indicating little to no cellular uptake.
Engineered nanobodies (pcNB1-GFP, Red/Green/Blue) were able to enter cells, and more of the protein entered as the dose increased. In contrast, the unmodified nanobody (NB1-GFP, Grey) and the fluorescent marker alone (GFP alone, Black) showed little to no ability to get inside the cells.

Benefits

  • Efficiently penetrates mammalian cell membranes
  • Direct access to intracellular therapeutic targets
  • Maintains structure, stability, and binding function
  • Adaptable scaffold for evolving new protein therapeutics
  • Compatible with high-throughput screening and protein engineering
  • Expands the range of druggable targets beyond the cell surface

Applications

  • Intracellular biologic drug discovery
  • Therapeutic development for cancer and other diseases
  • Protein-based research tools for studying cell pathways
  • Modulation or inhibition of intracellular proteins
  • Platform for next-generation biologic therapeutics

Publications

VJ Bruce, et al. (2016) Resurfaced cell-penetrating nanobodies: A potentially general scaffold for intracellularly targeted protein discovery. Protein Sci. doi: 10.1002/pro.2926.

 

Last Updated: March 2026
model of structure of the cell-penetrating nanobody construct
Opportunity

Available for Exclusive Licensing

IP Status

US 15398368

 

Inventors

Brian McNaughton
Virginia Bruce

Reference Number
16-046
Licensing Manager

Steve Foster
Steve.Foster@colostate.edu
970-491-7100

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