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Verma V, Sinha N, Raja A. Nanoscale warriors against viral invaders: a comprehensive review of Nanobodies as potential antiviral therapeutics. MAbs 2025; 17:2486390. [PMID: 40201976 PMCID: PMC11988260 DOI: 10.1080/19420862.2025.2486390] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2025] [Revised: 03/23/2025] [Accepted: 03/24/2025] [Indexed: 04/10/2025] Open
Abstract
Viral infections remain a significant global health threat, with emerging and reemerging viruses causing epidemics and pandemics. Despite advancements in antiviral therapies, the development of effective treatments is often hindered by challenges, such as viral resistance and the emergence of new strains. In this context, the development of novel therapeutic modalities is essential to combat notorious viruses. While traditional monoclonal antibodies are widely used for the treatment of several diseases, nanobodies derived from heavy chain-only antibodies have emerged as promising "nanoscale warriors" against viral infections. Nanobodies possess unique structural properties that enhance their ability to recognize diverse epitopes. Their small size also imparts properties, such as improved bioavailability, solubility, stability, and proteolytic resistance, making them an ideal class of therapeutics for viral infections. In this review, we discuss the role of nanobodies as antivirals against various viruses. Techniques used for developing nanobodies, delivery strategies are covered, and the challenges and opportunities associated with their use as antiviral therapies are discussed. We also offer insights into the future of nanobody-based antiviral research to support the development of new strategies for managing viral infections.
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Affiliation(s)
- Vaishali Verma
- Department of Biotechnology, School of Engineering and Applied Sciences, Bennett University, Greater Noida, India
| | - Nimisha Sinha
- Department of Biochemistry, Sri Venkateswara College, University of Delhi, New Delhi, India
| | - Abhavya Raja
- Department of Biotechnology, School of Engineering and Applied Sciences, Bennett University, Greater Noida, India
- Department of Surgery and Cancer, Imperial College London, South, London, UK
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2
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Rizk SS, Moustafa DM, ElBanna SA, Nour El-Din HT, Attia AS. Nanobodies in the fight against infectious diseases: repurposing nature's tiny weapons. World J Microbiol Biotechnol 2024; 40:209. [PMID: 38771414 PMCID: PMC11108896 DOI: 10.1007/s11274-024-03990-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2024] [Accepted: 04/15/2024] [Indexed: 05/22/2024]
Abstract
Nanobodies are the smallest known antigen-binding molecules to date. Their small size, good tissue penetration, high stability and solubility, ease of expression, refolding ability, and negligible immunogenicity in the human body have granted them excellence over conventional antibodies. Those exceptional attributes of nanobodies make them promising candidates for various applications in biotechnology, medicine, protein engineering, structural biology, food, and agriculture. This review presents an overview of their structure, development methods, advantages, possible challenges, and applications with special emphasis on infectious diseases-related ones. A showcase of how nanobodies can be harnessed for applications including neutralization of viruses and combating antibiotic-resistant bacteria is detailed. Overall, the impact of nanobodies in vaccine design, rapid diagnostics, and targeted therapies, besides exploring their role in deciphering microbial structures and virulence mechanisms are highlighted. Indeed, nanobodies are reshaping the future of infectious disease prevention and treatment.
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Affiliation(s)
- Soha S Rizk
- Microbiology and Immunology Postgraduate Program, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt
| | - Dina M Moustafa
- Department of Medical Sciences, Faculty of Dentistry, The British University in Egypt, El Sherouk City, Cairo, 11837, Egypt
| | - Shahira A ElBanna
- Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt
| | - Hanzada T Nour El-Din
- Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt
| | - Ahmed S Attia
- Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt.
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3
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Prado NDR, Brilhante-Da-Silva N, Sousa RMO, Morais MSDS, Roberto SA, Luiz MB, Assis LCD, Marinho ACM, Araujo LFLD, Pontes RDS, Stabeli RG, Fernandes CFC, Pereira SDS. Single-domain antibodies applied as antiviral immunotherapeutics. J Virol Methods 2023; 320:114787. [PMID: 37516366 DOI: 10.1016/j.jviromet.2023.114787] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Revised: 07/25/2023] [Accepted: 07/26/2023] [Indexed: 07/31/2023]
Abstract
Viral infections have been the cause of high mortality rates throughout different periods in history. Over the last two decades, outbreaks caused by zoonotic diseases and transmitted by arboviruses have had a significant impact on human health. The emergence of viral infections in different parts of the world encourages the search for new inputs to fight pathologies of viral origin. Antibodies represent the predominant class of new drugs developed in recent years and approved for the treatment of various human diseases, including cancer, autoimmune and infectious diseases. A promising group of antibodies are single-domain antibodies derived from camelid heavy chain immunoglobulins, or VHHs, are biomolecules with nanometric dimensions and unique pharmaceutical and biophysical properties that can be used in the diagnosis and immunotherapy of viral infections. For viral neutralization to occur, VHHs can act in different stages of the viral cycle, including the actual inhibition of infection, to hindering viral replication or assembly. This review article addresses advances involving the use of VHHs in therapeutic propositions aimed to battle different viruses that affect human health.
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Affiliation(s)
- Nidiane Dantas Reis Prado
- Laboratório de Engenharia de Anticorpos, Fundação Oswaldo Cruz, FIOCRUZ, unidade Rondônia, Porto Velho, RO, Brazil
| | - Nairo Brilhante-Da-Silva
- Laboratório de Engenharia de Anticorpos, Fundação Oswaldo Cruz, FIOCRUZ, unidade Rondônia, Porto Velho, RO, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto Oswaldo Cruz, IOC, Rio de Janeiro, RJ, Brazil
| | - Rosa Maria Oliveira Sousa
- Laboratório de Engenharia de Anticorpos, Fundação Oswaldo Cruz, FIOCRUZ, unidade Rondônia, Porto Velho, RO, Brazil
| | | | - Sibele Andrade Roberto
- Plataforma Bi-institucional de Medicina Translacional, Fundação Oswaldo Cruz-USP, Ribeirão Preto, SP, Brazil
| | - Marcos Barros Luiz
- Instituto Federal de Rondônia Campus Guajará-Mirim, IFRO, Guajará-Mirim, RO, Brazil
| | - Livia Coelho de Assis
- Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto Oswaldo Cruz, IOC, Rio de Janeiro, RJ, Brazil; Laboratório Multiusuário de Pesquisa e Desenvolvimento, Fundação Oswaldo Cruz, Fiocruz unidade Ceará, Eusebio, CE, Brazil
| | - Anna Carolina M Marinho
- Laboratório Multiusuário de Pesquisa e Desenvolvimento, Fundação Oswaldo Cruz, Fiocruz unidade Ceará, Eusebio, CE, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Ceará, Fortaleza, CE, Brazil
| | - Luiz Felipe Lemes de Araujo
- Plataforma Bi-institucional de Medicina Translacional, Fundação Oswaldo Cruz-USP, Ribeirão Preto, SP, Brazil; Programa de Pós-Graduação em Imunologia Básica e Aplicada, Universidade de São Paulo, USP, Ribeirão Preto, SP, Brazil
| | - Rafael de Souza Pontes
- Plataforma Bi-institucional de Medicina Translacional, Fundação Oswaldo Cruz-USP, Ribeirão Preto, SP, Brazil; Programa de Pós-Graduação em Imunologia Básica e Aplicada, Universidade de São Paulo, USP, Ribeirão Preto, SP, Brazil
| | - Rodrigo Guerino Stabeli
- Plataforma Bi-institucional de Medicina Translacional, Fundação Oswaldo Cruz-USP, Ribeirão Preto, SP, Brazil
| | - Carla Freire Celedonio Fernandes
- Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto Oswaldo Cruz, IOC, Rio de Janeiro, RJ, Brazil; Laboratório Multiusuário de Pesquisa e Desenvolvimento, Fundação Oswaldo Cruz, Fiocruz unidade Ceará, Eusebio, CE, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Ceará, Fortaleza, CE, Brazil
| | - Soraya Dos Santos Pereira
- Laboratório de Engenharia de Anticorpos, Fundação Oswaldo Cruz, FIOCRUZ, unidade Rondônia, Porto Velho, RO, Brazil; Programa de Pós-Graduação em Biologia Celular e Molecular, Instituto Oswaldo Cruz, IOC, Rio de Janeiro, RJ, Brazil; Programa de Pós-graduação em Biologia Experimental, Universidade Federal de Rondônia, UNIR, Porto Velho, RO, Brazil.
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Moliner-Morro A, McInerney GM, Hanke L. Nanobodies in the limelight: Multifunctional tools in the fight against viruses. J Gen Virol 2022; 103. [PMID: 35579613 DOI: 10.1099/jgv.0.001731] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Antibodies are natural antivirals generated by the vertebrate immune system in response to viral infection or vaccination. Unsurprisingly, they are also key molecules in the virologist's molecular toolbox. With new developments in methods for protein engineering, protein functionalization and application, smaller antibody-derived fragments are moving in focus. Among these, camelid-derived nanobodies play a prominent role. Nanobodies can replace full-sized antibodies in most applications and enable new possible applications for which conventional antibodies are challenging to use. Here we review the versatile nature of nanobodies, discuss their promise as antiviral therapeutics, for diagnostics, and their suitability as research tools to uncover novel aspects of viral infection and disease.
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Affiliation(s)
- Ainhoa Moliner-Morro
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
| | - Gerald M McInerney
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
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Wang Y, Mei Y, Ao Z, Chen Y, Jiang Y, Chen X, Qi R, Fu B, Tang J, Fang M, You M, Zhang T, Yuan Q, Luo W, Xia N. A broad-spectrum nanobody targeting the C-terminus of the hepatitis B surface antigen for chronic hepatitis B infection therapy. Antiviral Res 2022; 199:105265. [PMID: 35183645 DOI: 10.1016/j.antiviral.2022.105265] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Revised: 01/25/2022] [Accepted: 02/07/2022] [Indexed: 12/16/2022]
Abstract
Sustainable viral suppression with hepatitis B surface antigen (HBsAg) loss is the new treatment goal for chronic hepatitis B (CHB). The role of antibodies in therapies for persistent hepatitis B virus (HBV) infection has received constant attention. While immunotherapy holds great promise, challenges for the antibody-based prevention and control of HBV in CHB include broad HBV antigenic diversity and the need for long-term viral suppression. In this study, we identified a new anti-HBsAg nanobody (Nb), 125s, isolated from HBsAg-immunized alpaca and confirmed its excellent potency in HBsAg clearance and broad-spectrum therapeutic activity against three HBV subtypes in vivo. In addition, we characterized a novel epitope at the C-terminus of the HBsAg surface motif from amino acids 157 to 174. A 125s-based long-term passive immunization program was efficacious at HBsAg clearance and inducing cellular immune responses, offering a promising outlook for CHB immunotherapy.
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Affiliation(s)
- Yue Wang
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Yaxian Mei
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Zhenghong Ao
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Yuanzhi Chen
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Yichao Jiang
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Xiaoqing Chen
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Ruoyao Qi
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Baorong Fu
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Jixian Tang
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Mujin Fang
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Min You
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Tianying Zhang
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
| | - Quan Yuan
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China.
| | - Wenxin Luo
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China.
| | - Ningshao Xia
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, School of Life Science, Xiamen University, Xiamen, 361105, China
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Chen F, Liu Z, Jiang F. Prospects of Neutralizing Nanobodies Against SARS-CoV-2. Front Immunol 2021; 12:690742. [PMID: 34122456 PMCID: PMC8194341 DOI: 10.3389/fimmu.2021.690742] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2021] [Accepted: 05/04/2021] [Indexed: 01/14/2023] Open
Abstract
Since December 2019, the SARS-CoV-2 has erupted on a large scale worldwide and spread rapidly. Passive immunization of antibody-related molecules provides opportunities for prevention and treatment of high-risk patients and children. Nanobodies (Nbs) have many strong physical and chemical properties. They can be atomized, administered by inhalation, and can be directly applied to the infected site, with fast onset, high local drug concentration/high bioavailability, and high patient compliance (no needles). It has very attractive potential in the treatment of respiratory viruses. Rapid and low-cost development of Nbs targeting SARS-CoV-2 can quickly be achieved. Nbs against SARS-CoV-2 mutant strains also can be utilized quickly to prevent the virus from escaping. It provides important technical supports for the treatment of the SARS-CoV-2 and has the potential to become an essential medicine in the toolbox against the SARS-CoV-2.
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Affiliation(s)
- Fangfang Chen
- Department of Pharmacy, Shenzhen Hospital, Southern Medical University, Shenzhen, China
| | - Zhihong Liu
- State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
| | - Fan Jiang
- NanoAI Biotech Co., Ltd., Huahan Technology Industrial Park, Shenzhen, China
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Piramoon M, Khodadust F, Hosseinimehr SJ. Radiolabeled nanobodies for tumor targeting: From bioengineering to imaging and therapy. Biochim Biophys Acta Rev Cancer 2021; 1875:188529. [PMID: 33647388 DOI: 10.1016/j.bbcan.2021.188529] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2020] [Revised: 02/13/2021] [Accepted: 02/18/2021] [Indexed: 02/08/2023]
Abstract
So far, numerous molecules and biomolecules have been evaluated for tumor targeting purposes for radionuclide-based imaging and therapy modalities. Due to the high affinity and specificity against tumor antigens, monoclonal antibodies are appropriate candidates for tumor targeting. However, their large size prevents their comprehensive application in radionuclide-based tumor imaging or therapy, since it leads to their low tumor penetration, low blood clearance, and thus inappropriate tumor-to-background ratio. Nowadays, the variable domain of heavy-chain antibodies from the Camelidae family, known as nanobodies (Nbs), turn into exciting candidates for medical research. Considering several innate advantages of these new tumor-targeting agents, including excellent affinity and specificity toward antigen, high solubility, high stability, fast washout from blood, convenient production, ease of selection, and low immunogenicity, it assumes that they may overcome generic problems of monoclonal antibodies, their fragments, and other vectors used for tumor imaging/therapy. After three decades of Nbs discovery, the increasing number of their preclinical and clinical investigations, which have led to outstanding results, confirm their application for tumor targeting purposes. This review describes Nbs characteristics, the diagnostic and therapeutic application of their radioconjugates, and their recent advances.
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Affiliation(s)
- Majid Piramoon
- Department of Medicinal Chemistry and Radiopharmacy, School of Pharmacy, Lorestan University of Medical Sciences, Khorramabad, Iran; Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran
| | - Fatemeh Khodadust
- Department of Radiopharmacy, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran; Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran
| | - Seyed Jalal Hosseinimehr
- Department of Radiopharmacy, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran.
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Bessalah S, Jebahi S, Mejri N, Salhi I, Khorchani T, Hammadi M. Perspective on therapeutic and diagnostic potential of camel nanobodies for coronavirus disease-19 (COVID-19). 3 Biotech 2021; 11:89. [PMID: 33500874 PMCID: PMC7820838 DOI: 10.1007/s13205-021-02647-5] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Accepted: 01/06/2021] [Indexed: 12/11/2022] Open
Abstract
In this paper, we focus on the camelid nanobodies as a revolutionary therapy that can guide efforts to discover new drugs for Coronavirus disease (COVID-19). The small size property makes nanobodies capable of penetrating efficiently into tissues and recognizing cryptic antigens. Strong antigen affinity and stability in the gastrointestinal tract allow them to be used via oral administration. In fact, the use of nanobodies as inhalant can be directly delivered to the target organ, conferring high pulmonary drug concentrations and low systemic drug concentrations and minimal systemic side effects. For that, nanobodies are referred as a class of next-generation antibodies. Nanobodies permit the construction of multivalent formats that may achieve ultra-high neutralization potency and then may prevent mutational escape and can neutralize a wide range of SARS-CoV-2 variants. Due to their distinctive characteristics, nanobodies can be of great use in the development of promising treatment or preventive strategies against SARS-CoV-2 infection. In this review, the state-of-the-art of camel nanobodies design strategies against the virus including SARS-CoV-2 are critically summarized. The application of general nanotechnology was also discussed to mitigate and control emerging SARS-CoV-2 infection.
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Affiliation(s)
- Salma Bessalah
- Livestock and Wildlife Laboratory, Arid Lands Institute (I.R.A), University of Gabès, 4119 Médenine, Tunisia
| | - Samira Jebahi
- Laboratory on Energy and Matter for Nuclear Sciences Development (LR16CNSTN02), National Centre for Nuclear Sciences and Technologies, Sidi Thabet Technopark, 2020 Sidi Thabet, Tunisia, Pole technologique, BP 72, 2020 Sidi Thabet, Tunisia
| | - Naceur Mejri
- Laboratory on Energy and Matter for Nuclear Sciences Development (LR16CNSTN02), National Centre for Nuclear Sciences and Technologies, Sidi Thabet Technopark, 2020 Sidi Thabet, Tunisia, Pole technologique, BP 72, 2020 Sidi Thabet, Tunisia
| | - Imed Salhi
- Livestock and Wildlife Laboratory, Arid Lands Institute (I.R.A), University of Gabès, 4119 Médenine, Tunisia
| | - Touhami Khorchani
- Livestock and Wildlife Laboratory, Arid Lands Institute (I.R.A), University of Gabès, 4119 Médenine, Tunisia
| | - Mohamed Hammadi
- Livestock and Wildlife Laboratory, Arid Lands Institute (I.R.A), University of Gabès, 4119 Médenine, Tunisia
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Wagner TR, Rothbauer U. Nanobodies Right in the Middle: Intrabodies as Toolbox to Visualize and Modulate Antigens in the Living Cell. Biomolecules 2020; 10:biom10121701. [PMID: 33371447 PMCID: PMC7767433 DOI: 10.3390/biom10121701] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 12/15/2020] [Accepted: 12/18/2020] [Indexed: 01/01/2023] Open
Abstract
In biomedical research, there is an ongoing demand for new technologies to elucidate disease mechanisms and develop novel therapeutics. This requires comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, post-translational modifications and dynamic interactions of cellular components. Traceable intracellular binding molecules provide new opportunities for real-time cellular diagnostics. Most prominently, intrabodies derived from antibody fragments of heavy-chain only antibodies of camelids (nanobodies) have emerged as highly versatile and attractive probes to study and manipulate antigens within the context of living cells. In this review, we provide an overview on the selection, delivery and usage of intrabodies to visualize and monitor cellular antigens in living cells and organisms. Additionally, we summarize recent advances in the development of intrabodies as cellular biosensors and their application to manipulate disease-related cellular processes. Finally, we highlight switchable intrabodies, which open entirely new possibilities for real-time cell-based diagnostics including live-cell imaging, target validation and generation of precisely controllable binding reagents for future therapeutic applications.
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Affiliation(s)
- Teresa R. Wagner
- Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, 72076 Tuebingen, Germany;
- Natural and Medical Sciences Institute, University of Tuebingen, 72770 Reutlingen, Germany
| | - Ulrich Rothbauer
- Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, 72076 Tuebingen, Germany;
- Natural and Medical Sciences Institute, University of Tuebingen, 72770 Reutlingen, Germany
- Correspondence: ; Tel.: +49-7121-5153-0415; Fax: +49-7121-5153-0816
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Soetens E, Ballegeer M, Saelens X. An Inside Job: Applications of Intracellular Single Domain Antibodies. Biomolecules 2020; 10:biom10121663. [PMID: 33322697 PMCID: PMC7764588 DOI: 10.3390/biom10121663] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2020] [Revised: 12/09/2020] [Accepted: 12/10/2020] [Indexed: 02/06/2023] Open
Abstract
Sera of camelid species contain a special kind of antibody that consists only of heavy chains. The variable antigen binding domain of these heavy chain antibodies can be expressed as a separate entity, called a single domain antibody that is characterized by its small size, high solubility and oftentimes exceptional stability. Because of this, most single domain antibodies fold correctly when expressed in the reducing environment of the cytoplasm, and thereby retain their antigen binding specificity. Single domain antibodies can thus be used to target a broad range of intracellular proteins. Such intracellular single domain antibodies are also known as intrabodies, and have proven to be highly useful tools for basic research by allowing visualization, disruption and even targeted degradation of intracellular proteins. Furthermore, intrabodies can be used to uncover prospective new therapeutic targets and have the potential to be applied in therapeutic settings in the future. In this review we provide a brief overview of recent advances in the field of intracellular single domain antibodies, focusing on their use as research tools and potential therapeutic applications. Special attention is given to the available methods that allow delivery of single domain antibodies into cells.
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Affiliation(s)
- Eline Soetens
- VIB-UGent Center for Medical Biotechnology, VIB, B-9052 Ghent, Belgium; (E.S.); (M.B.)
- Department of Biochemistry and Microbiology, Ghent University, B-9000 Ghent, Belgium
| | - Marlies Ballegeer
- VIB-UGent Center for Medical Biotechnology, VIB, B-9052 Ghent, Belgium; (E.S.); (M.B.)
- Department of Biochemistry and Microbiology, Ghent University, B-9000 Ghent, Belgium
| | - Xavier Saelens
- VIB-UGent Center for Medical Biotechnology, VIB, B-9052 Ghent, Belgium; (E.S.); (M.B.)
- Department of Biochemistry and Microbiology, Ghent University, B-9000 Ghent, Belgium
- Correspondence:
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Zhang C, Ötjengerdes RM, Roewe J, Mejias R, Marschall ALJ. Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology. BioDrugs 2020; 34:435-462. [PMID: 32301049 PMCID: PMC7391400 DOI: 10.1007/s40259-020-00419-w] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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.
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Affiliation(s)
- Congcong Zhang
- Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany
- German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Frankfurt am Main, Germany
- German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Rina M Ötjengerdes
- Hannover Medical School (MHH), Carl-Neuberg-Straße 1, 30625, Hannover, Germany
| | - Julian Roewe
- German Cancer Consortium (DKTK) Clinical Cooperation Unit (CCU) Neuroimmunology and Brain TumorImmunology (D170), German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Rebeca Mejias
- School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK
| | - Andrea L J Marschall
- Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Brunswick, Germany.
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12
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Sanaei M, Setayesh N, Sepehrizadeh Z, Mahdavi M, Yazdi MH. Nanobodies in Human Infections: Prevention, Detection, and Treatment. Immunol Invest 2019; 49:875-896. [PMID: 31856615 DOI: 10.1080/08820139.2019.1688828] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Despite the existence of vaccination, antibiotic therapy, and antibody therapies, infectious diseases still remain as one of the biggest challenges to human health all over the world. Among the different methods for treatment and prevention of infectious diseases, antibodies are well known but poorly developed. There is a new subclass of antibodies calledheavy-chain antibodies that belong to the IgG isotype. However, they are low in molecular weight and lost the first constant domain (CH1). Their single-domain antigen-binding fragments, identified as nanobodies, have unique characteristics, which make them superior in comparison with the conventional antibodies. Low molecular weight and small size, high stability and solubility, ease of expression, good tissue penetration, and low-cost production make nanobodies an appropriate alternative to use against infectious disease. In this research, we review the properties of nanobodies and their potential applications in controlling human infections and inflammations.
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Affiliation(s)
- Marzieh Sanaei
- Biotechnology Research Center, Tehran University of Medical Sciences , Tehran, Iran.,Department of Pharmaceutical Biotechnology, Faculty of Pharmacy & Biotechnology Research Center, Tehran University of Medical Sciences , Tehran, Iran
| | - Neda Setayesh
- Department of Pharmaceutical Biotechnology, Faculty of Pharmacy & Biotechnology Research Center, Tehran University of Medical Sciences , Tehran, Iran
| | - Zargham Sepehrizadeh
- Department of Pharmaceutical Biotechnology, Faculty of Pharmacy & Biotechnology Research Center, Tehran University of Medical Sciences , Tehran, Iran
| | - Mehdi Mahdavi
- Recombinant Vaccine Research Center, Tehran University of Medical Sciences , Tehran, Iran
| | - Mohammad Hossein Yazdi
- Biotechnology Research Center, Tehran University of Medical Sciences , Tehran, Iran.,Recombinant Vaccine Research Center, Tehran University of Medical Sciences , Tehran, Iran
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13
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Chiu ML, Goulet DR, Teplyakov A, Gilliland GL. Antibody Structure and Function: The Basis for Engineering Therapeutics. Antibodies (Basel) 2019; 8:antib8040055. [PMID: 31816964 PMCID: PMC6963682 DOI: 10.3390/antib8040055] [Citation(s) in RCA: 294] [Impact Index Per Article: 49.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2019] [Revised: 11/25/2019] [Accepted: 11/28/2019] [Indexed: 12/11/2022] Open
Abstract
Antibodies and antibody-derived macromolecules have established themselves as the mainstay in protein-based therapeutic molecules (biologics). Our knowledge of the structure–function relationships of antibodies provides a platform for protein engineering that has been exploited to generate a wide range of biologics for a host of therapeutic indications. In this review, our basic understanding of the antibody structure is described along with how that knowledge has leveraged the engineering of antibody and antibody-related therapeutics having the appropriate antigen affinity, effector function, and biophysical properties. The platforms examined include the development of antibodies, antibody fragments, bispecific antibody, and antibody fusion products, whose efficacy and manufacturability can be improved via humanization, affinity modulation, and stability enhancement. We also review the design and selection of binding arms, and avidity modulation. Different strategies of preparing bispecific and multispecific molecules for an array of therapeutic applications are included.
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Affiliation(s)
- Mark L. Chiu
- Drug Product Development Science, Janssen Research & Development, LLC, Malvern, PA 19355, USA
- Correspondence:
| | - Dennis R. Goulet
- Department of Medicinal Chemistry, University of Washington, P.O. Box 357610, Seattle, WA 98195-7610, USA;
| | - Alexey Teplyakov
- Biologics Research, Janssen Research & Development, LLC, Spring House, PA 19477, USA; (A.T.); (G.L.G.)
| | - Gary L. Gilliland
- Biologics Research, Janssen Research & Development, LLC, Spring House, PA 19477, USA; (A.T.); (G.L.G.)
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14
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Single-Domain Antibodies Represent Novel Alternatives to Monoclonal Antibodies as Targeting Agents against the Human Papillomavirus 16 E6 Protein. Int J Mol Sci 2019; 20:ijms20092088. [PMID: 31035322 PMCID: PMC6539864 DOI: 10.3390/ijms20092088] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2019] [Revised: 04/23/2019] [Accepted: 04/26/2019] [Indexed: 01/18/2023] Open
Abstract
Approximately one fifth of all malignancies worldwide are etiologically associated with a persistent viral or bacterial infection. Thus, there is a particular interest in therapeutic molecules which use components of a natural immune response to specifically inhibit oncogenic microbial proteins, as it is anticipated they will elicit fewer off-target effects than conventional treatments. This concept has been explored in the context of human papillomavirus 16 (HPV16)-related cancers, through the development of monoclonal antibodies and fragments thereof against the viral E6 oncoprotein. Challenges related to the biology of E6 as well as the functional properties of the antibodies themselves appear to have precluded their clinical translation. Here, we addressed these issues by exploring the utility of the variable domains of camelid heavy-chain-only antibodies (denoted as VHHs). Through construction and panning of two llama, immune VHH phage display libraries, a pool of potential VHHs was isolated. The interactions of these with recombinant E6 were further characterized using an enzyme-linked immunosorbent assay (ELISA), Western blotting under denaturing and native conditions, and surface plasmon resonance. Three VHHs were identified that bound recombinant E6 with nanomolar affinities. Our results lead the way for subsequent studies into the ability of these novel molecules to inhibit HPV16-infected cells in vitro and in vivo.
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15
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Singh S, Murillo G, Chen D, Parihar AS, Mehta RG. Suppression of Breast Cancer Cell Proliferation by Selective Single-Domain Antibody for Intracellular STAT3. BREAST CANCER-BASIC AND CLINICAL RESEARCH 2018; 12:1178223417750858. [PMID: 29434474 PMCID: PMC5802608 DOI: 10.1177/1178223417750858] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/18/2017] [Accepted: 10/30/2017] [Indexed: 01/08/2023]
Abstract
Background: The serendipitous discovery of heavy-chain antibodies devoid of light chains in camelids and the subsequent development of VHHs (variable region of camelid heavy chain) have provided a very important tool for research and possibly for therapeutics. In this study, we synthesized single-domain 15-kDa antibody SBT-100 (anti-STAT3 B VHH13) against human STAT3 (signal transducer and activator of transcription) that binds selectively to STAT3 and suppresses the function of phosphorylated STAT3 (p-STAT3). Methods: Single-chain VHH nanobodies were generated by immunizing camelid with humanized STAT3. Commercially available breast cancer cell lines including MDA-MB-231, MDA-MB-468, MDA-MB-453, MCF-7, and BT474 were used. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The association of anti-STAT3 B VHH13 with STAT3 and p-STAT3 was determined by immunoprecipitation and Western blot analyses. The efficacy of SBT-100 on the growth of MDA-MB-231 xenografts in vivo was determined using athymic mice. Statistical significance for cell proliferation was determined using analysis of variance. If a significant difference (P < .05) was observed, then Tukey-Kramer multiple comparison test was conducted. Results: SBT-100 suppressed cell proliferation of triple-negative breast cancer cells (P < .01) as well as provided significant inhibition of tumor growth (P < .05) in a xenograft model without any toxicity. Results are presented to show that anti-STAT3 B VHH13 selectively binds to STAT3 suggesting that the effects were mediated by inhibiting STAT3. Conclusions: A very large number of human malignancies and benign diseases have constitutive STAT3 activation. Therefore, the results described here suggest that anti-STAT3 B VHH13 can be developed for therapeutic intervention for cancer cells expressing STAT3 or p-STAT3.
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Affiliation(s)
- Sunanda Singh
- Singh Biotechnology and Tampa Bay Technology Incubator, University of South Florida, Tampa Bay, FL, USA
| | | | | | - Ashutosh S Parihar
- Singh Biotechnology and Tampa Bay Technology Incubator, University of South Florida, Tampa Bay, FL, USA
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16
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Li Y, Liu Z, Hui L, Liu X, Feng A, Wang W, Zhang L, Li N, Zhou G, Wang Q, Han Q, Lv Y, Wang Q, Yang G, Wang Y. Transbody against virus core protein potently inhibits hepadnavirus replication in vivo: evidence from a duck model of hepatitis B virus. Br J Pharmacol 2017; 174:2261-2272. [PMID: 28383135 PMCID: PMC5481659 DOI: 10.1111/bph.13811] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2016] [Revised: 03/23/2017] [Accepted: 03/27/2017] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND AND PURPOSE The therapeutic management of hepatitis B virus (HBV) infections remains challenging, and novel antiviral strategies are urgently required. The HBV transbody, a monoclonal antibody (MAb) against human HBcAg coupled with the trans-activator of transcription protein transduction domain (TAT PTD), was previously shown to possess cell-penetrating ability and potent antiviral activity in vitro. The purpose of the present study was to evaluate the antiviral activity of the HBcMAb-TAT PTD conjugate in vivo in a duck model of HBV. EXPERIMENTAL APPROACH Female Peking ducks were injected i.p. with 0.03-0.3 mg·kg-1 ·day-1 of the DHBV transbody (DHBcMAb-TAT PTD conjugate) for 30 days. Serum DHBV DNA levels and liver DHBV DNA and covalently closed circular DNA (cccDNA) loads were determined at scheduled time points. Immunohistological examination of DHBcAg in the duck liver was also performed. KEY RESULTS The DHBV transbody significantly reduced the serum and liver DHBV DNA levels at doses of 0.1 and 0.3 mg·kg-1 ·day-1 and liver cccDNA levels at a dose of 0.3 mg·kg-1 ·day-1 after 30 days of treatment. The suppressive effects of the DHBV transbody at 0.3 mg·kg-1 ·day-1 on the serum and liver DHBV DNA and liver cccDNA levels remained significant, even at 15 days after treatment cessation. Similarly, immunohistochemistry of liver sections revealed decreased DHBcAg levels within hepatocytes 15 days after treatment termination. CONCLUSIONS AND IMPLICATIONS The DHBV transbody inhibits DHBV replication and possesses potent anti-DHBV activities in vivo. The cell-permeable antibody against the virus core antigen may be developed as a novel treatment for patients with hepadnavirus infections.
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Affiliation(s)
- Yiping Li
- School of PharmacyXi'an Jiaotong UniversityXi'anChina
| | - Zhengwen Liu
- Department of Infectious Diseases, First Affiliated Hospital, School of MedicineXi'an Jiaotong UniversityXi'anChina
- Institute of Advanced Surgical Technology and EngineeringXi'an Jiaotong UniversityXi'anChina
| | - Lingyun Hui
- Department of Laboratory Medicine, First Affiliated Hospital, School of medicineXi'an Jiaotong UniversityXi'anChina
| | - Xi Liu
- Department of Pathology, First Affiliated Hospital, School of medicineXi'an Jiaotong UniversityXi'anChina
| | - Ai Feng
- Department of Laboratory Medicine, First Affiliated Hospital, School of medicineXi'an Jiaotong UniversityXi'anChina
| | - Wei Wang
- Department of Laboratory Medicine, First Affiliated Hospital, School of medicineXi'an Jiaotong UniversityXi'anChina
| | - Lin Zhang
- Department of Laboratory Medicine, First Affiliated Hospital, School of medicineXi'an Jiaotong UniversityXi'anChina
| | - Na Li
- Department of Infectious Diseases, First Affiliated Hospital, School of MedicineXi'an Jiaotong UniversityXi'anChina
| | - Guoqing Zhou
- School of Computer Science and EngineeringNorthwestern Polytechnical UniversityXi'anChina
| | - Quanli Wang
- Department of Epidemiology and Statistics, School of medicineXi'an Jiaotong UniversityXi'anChina
| | - Qunying Han
- Department of Infectious Diseases, First Affiliated Hospital, School of MedicineXi'an Jiaotong UniversityXi'anChina
| | - Yi Lv
- Institute of Advanced Surgical Technology and EngineeringXi'an Jiaotong UniversityXi'anChina
- Department of Hepatobiliary Surgery, First Affiliated Hospital, School of MedicineXi'an Jiaotong UniversityXi'anChina
| | - Quanying Wang
- Xi'an Hua Guang Biological Engineering CompanyXi'anChina
| | - Guangxiao Yang
- Xi'an Hua Guang Biological Engineering CompanyXi'anChina
| | - Yawen Wang
- Department of Laboratory Medicine, First Affiliated Hospital, School of medicineXi'an Jiaotong UniversityXi'anChina
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17
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Fernandes CFC, Pereira SDS, Luiz MB, Zuliani JP, Furtado GP, Stabeli RG. Camelid Single-Domain Antibodies As an Alternative to Overcome Challenges Related to the Prevention, Detection, and Control of Neglected Tropical Diseases. Front Immunol 2017. [PMID: 28649245 PMCID: PMC5465246 DOI: 10.3389/fimmu.2017.00653] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Due mainly to properties such as high affinity and antigen specificity, antibodies have become important tools for biomedical research, diagnosis, and treatment of several human diseases. When the objective is to administer them for therapy, strategies are used to reduce the heterologous protein immunogenicity and to improve pharmacokinetic and pharmacodynamic characteristics. Size minimization contributes to ameliorate these characteristics, while preserving the antigen-antibody interaction site. Since the discovery that camelids produce functional antibodies devoid of light chains, studies have proposed the use of single domains for biosensors, monitoring and treatment of tumors, therapies for inflammatory and neurodegenerative diseases, drug delivery, or passive immunotherapy. Despite an expected increase in antibody and related products in the pharmaceutical market over the next years, few research initiatives are related to the development of alternatives for helping to manage neglected tropical diseases (NTDs). In this review, we summarize developments of camelid single-domain antibodies (VHH) in the field of NTDs. Particular attention is given to VHH-derived products, i.e., VHHs fused to nanoparticles, constructed for the development of rapid diagnostic kits; fused to oligomeric matrix proteins for viral neutralization; and conjugated with proteins for the treatment of human parasites. Moreover, paratransgenesis technology using VHHs is an interesting approach to control parasite development in vectors. With enormous biotechnological versatility, facility and low cost for heterologous production, and greater ability to recognize different epitopes, VHHs have appeared as an opportunity to overcome challenges related to the prevention, detection, and control of human diseases, especially NTDs.
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Affiliation(s)
| | | | - Marcos B Luiz
- Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, Rondônia, Brazil
| | - Juliana P Zuliani
- Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, Rondônia, Brazil.,Departamento de Medicina da Universidade Federal de Rondônia, UNIR, Porto Velho, Rondônia, Brazil
| | | | - Rodrigo G Stabeli
- Departamento de Medicina da Universidade Federal de Rondônia, UNIR, Porto Velho, Rondônia, Brazil.,Plataforma Bi-Institucional de Medicina Translacional (Fiocruz-USP), Ribeirão Preto, São Paulo, Brazil
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18
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Böldicke T. Single domain antibodies for the knockdown of cytosolic and nuclear proteins. Protein Sci 2017; 26:925-945. [PMID: 28271570 PMCID: PMC5405437 DOI: 10.1002/pro.3154] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2017] [Accepted: 03/03/2017] [Indexed: 12/12/2022]
Abstract
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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Affiliation(s)
- Thomas Böldicke
- Helmholtz Centre for Infection Research, Structure and Function of ProteinsInhoffenstraße 7, D‐38124BraunschweigGermany
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19
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Ubah O, Palliyil S. Monoclonal Antibodies and Antibody Like Fragments Derived from Immunised Phage Display Libraries. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 1053:99-117. [PMID: 29549637 PMCID: PMC7120432 DOI: 10.1007/978-3-319-72077-7_6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Morbidity and mortality associated with infectious diseases are always on the rise, especially in poorer countries and in the aging population. The inevitable, but unpredictable emergence of new infectious diseases has become a global threat. HIV/AIDS, severe acute respiratory syndrome (SARS), and the more recent H1N1 influenza are only a few of the numerous examples of emerging infectious diseases in the modern era. However despite advances in diagnostics, therapeutics and vaccines, there is need for more specific, efficacious, cost-effective and less toxic treatment and preventive drugs. In this chapter, we discuss a powerful combinatorial technology in association with animal immunisation that is capable of generating biologic drugs with high affinity, efficacy and limited off-site toxicity, and diagnostic tools with great precision. Although time consuming, immunisation still remains the preferred route for the isolation of high-affinity antibodies and antibody-like fragments. Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. The selection of binding fragments from phage display libraries has proven significant for routine isolation of invaluable peptides, antibodies, and antibody-like domains for diagnostic and therapeutic applications. Here we highlight the many benefits of combining immunisation with phage display in combating infectious diseases, and how our knowledge of antibody engineering has played a crucial role in fully exploiting these platforms in generating therapeutic and diagnostic biologics towards antigenic targets of infectious organisms.
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Affiliation(s)
- Obinna Ubah
- Scottish Biologics Facility, Elasmogen Ltd, Aberdeen, UK
| | - Soumya Palliyil
- Scottish Biologics Facility, University of Aberdeen, Aberdeen, UK.
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20
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Controlling Rotavirus-associated diarrhea: Could single-domain antibody fragments make the difference? Rev Argent Microbiol 2015; 47:368-79. [PMID: 26654700 DOI: 10.1016/j.ram.2015.09.005] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2015] [Revised: 09/15/2015] [Accepted: 09/21/2015] [Indexed: 12/30/2022] Open
Abstract
Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects.
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21
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de Marco A. Recombinant antibody production evolves into multiple options aimed at yielding reagents suitable for application-specific needs. Microb Cell Fact 2015; 14:125. [PMID: 26330219 PMCID: PMC4557595 DOI: 10.1186/s12934-015-0320-7] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2015] [Accepted: 08/20/2015] [Indexed: 01/02/2023] Open
Abstract
BACKGROUND Antibodies have been a pillar of basic research, while their relevance in clinical diagnostics and therapy is constantly growing. Consequently, the production of both conventional and fragment antibodies constantly faces more demanding challenges for the improvement of their quantity and quality. The answer to such an increasing need has been the development of a wide array of formats and alternative production platforms. This review offers a critical comparison and evaluation of the different options to help the researchers interested in expressing recombinant antibodies in their choice. RESULTS Rather than the compilation of an exhaustive list of the recent publications in the field, this review intendeds to analyze the development of the most innovative or fast-growing strategies. These have been illustrated with some significant examples and, when possible, compared with the existing alternatives. Space has also been given to those solutions that might represent interesting opportunities or that investigate critical aspects of the production optimization but for which the available data as yet do not allow for a definitive judgment. CONCLUSIONS The take-home message is that there is a clear process of progressive diversification concerning the antibody expression platforms and an effort to yield directly application-adapted immune-reagents rather than generic naked antibodies that need further in vitro modification steps before becoming usable.
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Affiliation(s)
- Ario de Marco
- Department of Biomedical Sciences and Engineering, University of Nova Gorica, Glavni Trg 9, 5261, Vipava, Slovenia.
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22
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Marschall ALJ, Dübel S, Böldicke T. Specific in vivo knockdown of protein function by intrabodies. MAbs 2015; 7:1010-35. [PMID: 26252565 PMCID: PMC4966517 DOI: 10.1080/19420862.2015.1076601] [Citation(s) in RCA: 78] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2015] [Revised: 07/19/2015] [Accepted: 07/20/2015] [Indexed: 01/02/2023] Open
Abstract
Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed.
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Affiliation(s)
- Andrea LJ Marschall
- Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics; Braunschweig, Germany
| | - Stefan Dübel
- Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics; Braunschweig, Germany
| | - Thomas Böldicke
- Helmholtz Centre for Infection Research, Recombinant Protein Expression/Intrabody Unit, Helmholtz Centre for Infection Research; Braunschweig, Germany
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23
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Wang Y, Li Y, Li N, Zhu Q, Hui L, Liu X, Han Q, Lv Y, Wang Q, Yang G, Zhou Z, Liu Z. Transbody against hepatitis B virus core protein inhibits hepatitis B virus replication in vitro. Int Immunopharmacol 2015; 25:363-369. [PMID: 25676532 DOI: 10.1016/j.intimp.2015.01.028] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2014] [Revised: 01/14/2015] [Accepted: 01/29/2015] [Indexed: 02/08/2023]
Abstract
Hepatitis B virus (HBV) infection is one of the major causes of chronic liver diseases. The current therapeutics show limited efficacy. In the HBV life cycle, virus core antigen (HBcAg) plays important multiple roles. Blocking the pleiotropic functions of HBcAg may thus represent a promising strategy for anti-HBV replication. In this study, monoclonal antibody (MAb) against core antigen of human HBV was coupled with TAT protein transduction domain (TAT PTD) to form transbody, and the effect on virus replication was evaluated in vitro. The HBV transbody, HBcMAb-TAT PTD conjugate, recognized HBcAg and retained cell-penetrating activity in living cells. In HBV-transfected liver cell line HepG2.2.15, HBV transbody suppressed not only the extracellular HBsAg, HBeAg and HBV DNA, but also the intracellular HBsAg, HBeAg, HBcAg and HBV DNA in a dose-dependent manner. These results indicate that the transbody prepared possesses readily cell-penetrating ability and potent antiviral activity, providing a novel approach, a cell-permeable antibody against HBcAg, for the treatment of HBV infection.
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Affiliation(s)
- Yawen Wang
- Department of Laboratory Medicine, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Yiping Li
- School of Pharmacy, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Na Li
- Department of Infectious Diseases, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Qianqian Zhu
- Department of Infectious Diseases, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Lingyun Hui
- Department of Laboratory Medicine, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Xi Liu
- Department of Pathology, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Qunying Han
- Department of Infectious Diseases, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Yi Lv
- Department of Hepatobiliary Surgery, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China; Institute of Advanced Surgical Technology and Engineering, Xi'an Jiaotong University, Xi'an, 710061, Shaanxi, China
| | - Quanying Wang
- Xi'an Hua Guang Biological Engineering Company, Xi'an, Shaanxi, China
| | - Guangxiao Yang
- Xi'an Hua Guang Biological Engineering Company, Xi'an, Shaanxi, China
| | - Zhihua Zhou
- Department of Infectious Diseases, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Zhengwen Liu
- Department of Infectious Diseases, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China; Institute of Advanced Surgical Technology and Engineering, Xi'an Jiaotong University, Xi'an, 710061, Shaanxi, China.
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Tan WS, Ho KL. Phage display creates innovative applications to combat hepatitis B virus. World J Gastroenterol 2014; 20:11650-11670. [PMID: 25206271 PMCID: PMC4155357 DOI: 10.3748/wjg.v20.i33.11650] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/25/2013] [Accepted: 05/05/2014] [Indexed: 02/06/2023] Open
Abstract
Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20th century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases.
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25
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Goldman ER, Brozozog-Lee PA, Zabetakis D, Turner KB, Walper SA, Liu JL, Anderson GP. Negative tail fusions can improve ruggedness of single domain antibodies. Protein Expr Purif 2014; 95:226-32. [PMID: 24440507 DOI: 10.1016/j.pep.2014.01.003] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2013] [Revised: 01/06/2014] [Accepted: 01/08/2014] [Indexed: 12/31/2022]
Abstract
Single-domain antibodies (sdAbs), the recombinantly expressed binding domains derived from the heavy-chain-only antibodies found in camelids and sharks, are valued for their ability to refold after heat denaturation. However, some sdAbs are prone to aggregation on extended heating at high concentration. Additionally, sdAbs prepared cytoplasmically often lack the conserved disulfide bond found in variable heavy domains, which both decreases their melting point and can decrease their ability to refold. Genetic fusions of sdAbs with the acid tail of α-synuclein (ATS) resulted in constructs that had enhanced ability to resist aggregation. In addition, almost complete refolding was observed even in the absence of the disulfide bond. These sdAb-ATS fusions expand the utility of sdAbs. They provide sdAbs that are resistant to aggregation, and enable the production of re-foldable sdAbs in the reducing environment of the cytoplasm.
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Affiliation(s)
- Ellen R Goldman
- Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA
| | | | - Dan Zabetakis
- Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA
| | - Kendrick B Turner
- Science and Engineering Apprenticeship Program, American Society for Engineering Education, 4555 Overlook Ave. SW, Washington, DC 20375, USA
| | - Scott A Walper
- Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA
| | - Jinny L Liu
- Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA
| | - George P Anderson
- Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375, USA.
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26
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Hassanzadeh-Ghassabeh G, Devoogdt N, De Pauw P, Vincke C, Muyldermans S. Nanobodies and their potential applications. Nanomedicine (Lond) 2013; 8:1013-26. [PMID: 23730699 DOI: 10.2217/nnm.13.86] [Citation(s) in RCA: 236] [Impact Index Per Article: 19.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Nanobodies are recombinant, antigen-specific, single-domain, variable fragments of camelid heavy chain-only antibodies. The innate supremacy of nanobodies as a renewable source of affinity reagents, together with their high production yield in a broad variety of expression systems, minimal size, great stability, reversible refolding and outstanding solubility in aqueous solutions, and ability to specifically recognize unique epitopes with subnanomolar affinity, have combined to make them a useful class of biomolecules for research and various medical diagnostic and therapeutic applications. This article speculates on a number of technological innovations that might be introduced in the nanobody identification platform to streamline the generation of more potent nanobodies and to expand their application range.
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Vanlandschoot P, Stortelers C, Beirnaert E, Ibañez LI, Schepens B, Depla E, Saelens X. Nanobodies®: New ammunition to battle viruses. Antiviral Res 2011; 92:389-407. [DOI: 10.1016/j.antiviral.2011.09.002] [Citation(s) in RCA: 83] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2011] [Revised: 08/30/2011] [Accepted: 09/06/2011] [Indexed: 01/23/2023]
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Govaert J, Pellis M, Deschacht N, Vincke C, Conrath K, Muyldermans S, Saerens D. Dual beneficial effect of interloop disulfide bond for single domain antibody fragments. J Biol Chem 2011; 287:1970-9. [PMID: 22128183 DOI: 10.1074/jbc.m111.242818] [Citation(s) in RCA: 114] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond.
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Affiliation(s)
- Jochen Govaert
- Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
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29
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de Marco A. Biotechnological applications of recombinant single-domain antibody fragments. Microb Cell Fact 2011; 10:44. [PMID: 21658216 PMCID: PMC3123181 DOI: 10.1186/1475-2859-10-44] [Citation(s) in RCA: 125] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2011] [Accepted: 06/09/2011] [Indexed: 01/29/2023] Open
Abstract
BACKGROUND Single-domain antibody fragments possess structural features, such as a small dimension, an elevated stability, and the singularity of recognizing epitopes non-accessible for conventional antibodies that make them interesting for several research and biotechnological applications. RESULTS The discovery of the single-domain antibody's potentials has stimulated their use in an increasing variety of fields. The rapid accumulation of articles describing new applications and further developments of established approaches has made it, therefore, necessary to update the previous reviews with a new and more complete summary of the topic. CONCLUSIONS Beside the necessary task of updating, this work analyses in detail some applicative aspects of the single-domain antibodies that have been overseen in the past, such as their efficacy in affinity chromatography, as co-crystallization chaperones, protein aggregation controllers, enzyme activity tuners, and the specificities of the unconventional single-domain fragments.
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Affiliation(s)
- Ario de Marco
- University of Nova Gorica (UNG), Vipavska 13, PO Box 301-SI-5000, Rožna Dolina (Nova Gorica), Slovenia.
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30
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Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody. Virology 2011; 411:132-41. [PMID: 21239030 DOI: 10.1016/j.virol.2010.12.034] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2010] [Revised: 10/27/2010] [Accepted: 12/20/2010] [Indexed: 12/18/2022]
Abstract
The Hepatitis B virus precore protein is processed in the endoplasmic reticulum (ER) into secreted hepatitis B e antigen (HBeAg), which acts as an immune tolerogen to establish chronic infection. Downregulation of secreted HBeAg should improve clinical outcome, as patients who effectively respond to current treatments (IFN-α) have significantly lower serum HBeAg levels. Here, we describe a novel reagent, a single variable domain (V(NAR)) of the shark immunoglobulin new antigen receptor (IgNAR) antibodies. V(NAR)s possess advantages in stability, size (~14 kDa) and cryptic epitope recognition compared to conventional antibodies. The V(NAR) domain displayed biologically useful affinity for recombinant and native HBeAg, and recognised a unique conformational epitope. To assess therapeutic potential in targeting intracellular precore protein to reduce secreted HBeAg, the V(NAR) was engineered for ER-targeted in vitro delivery to function as an intracellular antibody (intrabody). In vitro data from HBV/precore hepatocyte cell lines demonstrated effective intrabody regulation of precore/HBeAg.
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Intrabody Expression in Mammalian Cells. ANTIBODY EXPRESSION AND PRODUCTION 2011. [PMCID: PMC7120103 DOI: 10.1007/978-94-007-1257-7_9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 10/30/2022]
Abstract
The intracellular expression of antibodies or antibody fragments (intrabodies) in different compartments of mammalian cells allows to block or modulate the function of endogenous molecules. Intrabodies can alter protein folding, protein-protein, protein-DNA, protein-RNA interactions and protein modification. They can induce a phenotypic knockout and work as neutralizing agents by direct binding to the target antigen, by diverting its intracellular traffic or by inhibiting its association with binding partners. They have been largely employed as research tools and are emerging as therapeutic molecules for the treatment of human diseases as viral pathologies, cancer and misfolding diseases. The fast growing bio-market of recombinant antibodies provides intrabodies with enhanced binding specificity, stability and solubility, together with lower immunogenicity, for their use in therapy. This chapter describes the crucial aspects required to express intrabodies in different intracellular compartments of mammalian cells, their various modes of action and gives an update on the applications of intrabodies in human diseases.
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