Recent Advances with ER Targeted Intrabodies

Marschall, Andrea L. J.; Dübel, Stefan; Böldicke, Thomas

doi:10.1007/978-3-319-22473-2_5

Cited by 6 publications

(9 citation statements)

References 86 publications

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“…The ER intrabody approach has already been successfully applied in many research areas. Examples of the application of ER-retained intrabodies with a focus on methodological and technological aspects have been reviewed previously [ 22 ], and their therapeutic potential has been briefly discussed [ 59 , 60 ].…”

Section: Strategies To Use Antibodies Inside Living Cellsmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Strategies To Use Antibodies Inside Living Cellsmentioning

confidence: 99%

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

et al. 2020

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“…ER-intrabody knockdown function is based on retention of molecules passing the secretory pathway via the KDEL sequence fused to the C-terminal end of the intrabody. 6,[83][84][85] In the past ER-intrabodies have been the most promising molecules because they are correctly folded in the ER environment. 86 Contrary to ER intrabodies which have only to bind to the antigen inside the ER, cytosolic/nuclear intrabodies have to inactivate their targets by binding to the antigen and inducing a conformational change or interfering with the binding of the target protein to its specific binding partner.…”

Section: Er Intrabodies Versus Cytosolic/nuclear Intrabodiesmentioning

confidence: 99%

“…ER‐intrabody knockdown function is based on retention of molecules passing the secretory pathway via the KDEL sequence fused to the C‐terminal end of the intrabody . In the past ER‐intrabodies have been the most promising molecules because they are correctly folded in the ER environment .…”

Section: Introductionmentioning

confidence: 99%

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

Böldicke

2017

Protein Science

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Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.

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“…In vitro , there are already many examples for successful intrabody mediated knockdowns, both achieved with cytosolic intrabodies and ER intrabodies. Targets that have been knocked down by intrabodies include oncogenic targets, proteins related to immune function, neuronal targets including those involved in neurodegenerative disorders and targets involved in chronic viral infections, showing the broad range of potential application of future therapeutic intrabodies [31] . A comprehensive overview is given in Ref.…”

Section: Therapeutic Intrabodies Need Gene Therapymentioning

confidence: 99%

“…Therapeutic strategies against chronic viral infections include intrabodies targeted directly against viral proteins or intrabodies that target host proteins [31] . An intrabody targeting the host protein CCR5 that is involved in viral entry of HIV into host cells, has for instance been found to protect cells from infection [43] , [47] .…”

Section: Therapeutic Intrabodies Need Gene Therapymentioning

confidence: 99%

Antibodies inside of a cell can change its outside: Can intrabodies provide a new therapeutic paradigm?

Marschall

Dübel

2016

Computational and Structural Biotechnology Journal

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Challenges posed by complex diseases such as cancer, chronic viral infections, neurodegenerative disorders and many others have forced researchers to think beyond classic small molecule drugs, exploring new therapeutic strategies such as therapy with RNAi, CRISPR/Cas9 or antibody therapies as single or as combination therapies with existing drugs. While classic antibody therapies based on parenteral application can only reach extracellular targets, intracellular application of antibodies could provide specific advantages but is so far little recognized in translational research. Intrabodies allow high specificity and targeting of splice variants or post translational modifications. At the same time off target effects can be minimized by thorough biochemical characterization. Knockdown of cellular proteins by intrabodies has been reported for a significant number of disease-relevant targets, including ErbB-2, EGFR, VEGFR-2, Metalloproteinase MMP2 and MMP9, β-amyloid protein, α-synuclein, HIV gp120, HCV core and many others. This review outlines the recent advances in ER intrabody technology and their potential use in therapy.

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Recent Advances with ER Targeted Intrabodies

Cited by 6 publications

References 86 publications

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

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

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

Antibodies inside of a cell can change its outside: Can intrabodies provide a new therapeutic paradigm?

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