Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

Abstract

To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy. Proteins can be targeted directly and very specifically by antibodies, but using antibodies inside cells has so far been considered to be challenging. However, it is possible to deliver antibodies or other proteins into the cytosol using standard laboratory equipment. Physical methods such as electroporation have been demonstrated to be efficient and validated thoroughly over time. The expression of intracellular antibodies (intrabodies) inside cells is another way to interfere with intracellular targets at the protein level. Methodological strategies to target the inside of cells with antibodies, including delivered antibodies and expressed antibodies, as well as applications in the research areas of neurobiology, viral infections and oncology, are reviewed here. Antibodies have already been used to interfere with a wide range of intracellular targets. Disease-related targets included proteins associated with neurodegenerative diseases such as Parkinson's disease (α-synuclein), Alzheimer's disease (amyloid-β) or Huntington's disease (mutant huntingtin [mHtt]). The applications of intrabodies in the context of viral infections include targeting proteins associated with HIV (e.g. HIV1-TAT, Rev, Vif, gp41, gp120, gp160) and different oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Epstein-Barr virus, and they have been used to interfere with various targets related to different processes in cancer, including oncogenic pathways, proliferation, cell cycle, apoptosis, metastasis, angiogenesis or neo-antigens (e.g. p53, human epidermal growth factor receptor-2 [HER2], signal transducer and activator of transcription 3 [STAT3], RAS-related RHO-GTPase B (RHOB), cortactin, vascular endothelial growth factor receptor 2 [VEGFR2], Ras, Bcr-Abl). Interfering at the protein level allows questions to be addressed that may remain unanswered using alternative methods. This review addresses why direct targeting of proteins allows unique insights, what is currently feasible in vitro, and how this relates to potential therapeutic applications.Therapeutic antibodies are valuable drugs, which mostly act outside of cells.Reaching the numerous drug targets that reside inside cells by antibodies is possible in vitro and allows unique insights compared with other methods.Applying antibodies inside cells for therapeutic purposes has been explored in animal models and promises specific therapeutic benefits in neurobiology, virology and oncology.

Intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for therapy of established HPV-associated tumors

“…In this study, intrabodies were expressed directly in the target tumor cells by electroporating tumors with scFv-expressing recombinant plasmids. However, direct delivery of scFvs as proteins is also feasible [ 28 ].…”

Transforming nanobodies into high-precision tools for protein function analysis

“…Antibodies can be generated fairly rapidly once the protein (domain) under study can be obtained in suitable (recombinant) format for immunization. Single-chain variable fragments (scFvs) and nanobodies have been used to this end, but also synthetic alternatives are useful such as designed ankyrin repeat proteins (DARPins), affibodies (derived from protein A) or fibronectin folds (monobodies) (96,113,187).…”

Targeting Human Papillomavirus-Associated Cancer by Oncoprotein-Specific Recombinant Antibodies

“…Antibody engineering also allows cloning of scFv sequences into eukaryotic vectors equipped with intracellular localization signals, for the scFv expression in specific cell compartments as intrabodies. These can reach and recognize target antigens in the cellular compartments where they are located, with outcomes ranging from direct anti-gen blockade to indirect impairment of its activity through delocalization, to its targeted degradation [8,9].…”