Effective capture of proteins inside living cells by antibodies indirectly linked to a novel CPP-pAd

Developers

S. Itakura, S. Hama, K. Kogure etc.

Description of the technology

Antibodies against cytoplasmic proteins are useful tools that can control cellular function and clarify signaling mechanisms. However, it is difficult to capture proteins inside living cells, and thus appropriate methods for antibody delivery to the cytoplasm of living cells are required. Cell-penetrating materials, such as the TAT-peptide, have received attention for their ability to deliver various cargos into living cells. However, the direct modification of cargos with cell-penetrating materials is time-consuming and lacks versatility. Therefore, scientists conceived that protein A, which can bind to the fragment crystallizable region of an antibody, could indirectly link antibodies with cell-penetrating materials, creating an efficient and simple antibody delivery system. Here, we constructed a novel antibody delivery system using a cell-penetrating polymer-modified protein A derivative (CPP-pAd). Living cells treated with CPP-pAd/antibody complexes showed significantly higher antibody levels than those achieved with the commercially available reagent HVJ-E. Pre-treatment with sucrose prevented cellular uptake of the CPP-pAd/antibody complex, suggesting that the CPP-pAd/antibody internalization mechanism occurs through clathrin-dependent endocytosis. Interestingly, intracellularly delivered antibodies did not colocalize with endosome/lysosome markers, further suggesting that antibodies were delivered to the cytoplasm by escape from endosome/lysosome. Moreover, it was observed that anti-nuclear pore complex antibodies, delivered to cells using CPP-pAd, localized to the nuclear membrane and inhibited nuclear factor κB dependent luciferase activity.

Practical application

CPP-pAd use is a promising strategy for effective capture of proteins inside living cells.

Laboratories

• Department of Biophysical Chemistry, Kyoto Pharmaceutical University, Kyoto, (Japan)
• Institute for Chemical Research, Kyoto University, Kyoto, (Japan)
• Welsh School of Pharmacy, Cardiff University, Cardiff, Wales, (United Kingdom)

Links

http://www.ncbi.nlm.nih.gov/pubmed/25315678
https://books.google.com.ua/books?hl=ru&lr=&id=d4PXz6-gkO0C&oi=fnd&pg=PA263&dq=CPP-pAd&ots=mairU-3Qw...

Publications

• Itakura, Shoko, et al. "Effective capture of proteins inside living cells by antibodies indirectly linked to a novel cell‐penetrating polymer‐modified protein A derivative." FEBS Journal 282.1 (2015): 142-152.
• Takayama, Kentaro, et al. "Effect of the attachment of a penetration accelerating sequence and the influence of hydrophobicity on octaarginine-mediated intracellular delivery." Molecular pharmaceutics 9.5 (2012): 1222-1230.
• Watkins, Catherine L., et al. "Cellular uptake, distribution and cytotoxicity of the hydrophobic cell penetrating peptide sequence PFVYLI linked to the proapoptotic domain peptide PAD." Journal of Controlled Release 140.3 (2009): 237-244.