Journal articles: 'Nanobodies'

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Gibbs, W. Wayt. "Nanobodies." Scientific American 293, no. 2 (August 2005): 78–83. http://dx.doi.org/10.1038/scientificamerican0805-78.

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Weiss and Verrips. "Nanobodies that Neutralize HIV." Vaccines 7, no. 3 (July 31, 2019): 77. http://dx.doi.org/10.3390/vaccines7030077.

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Nanobodies or VHH (variable domains of heavy-chain only antibodies) are derived from camelid species such as llamas and camels. Nanobodies isolated and selected through phage display can neutralize a broad range of human immunodeficiency virus type 1 (HIV-1) strains. Nanobodies fit into canyons on the HIV envelope that may not be accessible to IgG (immunoglobulin G) containing both heavy and light chains, and they tend to have long CDR3 (complementarity-determining region 3) loops that further enhance recognition of otherwise cryptic epitopes. Nanobodies are readily expressed at high levels in bacteria and yeast, as well as by viral vectors, and they form relatively stable, heat-resistant molecules. Nanobodies can be linked to human Fc chains to gain immune effector functions. Bivalent and trivalent nanobodies recognizing the same or distinct epitopes on the envelope glycoproteins, gp120 and gp41, greatly increase the potency of HIV-1 neutralization. Nanobodies have potential applications for HIV-1 diagnostics, vaccine design, microbicides, immunoprophylaxis, and immunotherapy.

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Shatalova, A. V., A. S. Yakubova, V. V. Palimpsestov, and I. B. Esmagambetov. "NANOBODIES: STRUCTURE, MANUFACTURING, APPLICATION (REVIEW)." Drug development & registration 8, no. 1 (February 14, 2019): 14–22. http://dx.doi.org/10.33380/2305-2066-2019-8-1-14-22.

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Introduction. Single-domain antibodies (nanobodies) are composed of the heavy-chain variable domain only. Compared to conventional immunoglobulins G (IgG) nanobodies have such qualities as: high bioavailability, ability to bind epitopes that are difficult to reach, high solubility and thermal stability, etc. Nanobodies can be easily manufactured in microorganisms (E. coli) to significantly save on cost. Text. Goal of the paper consists of the description of structural and functional properties of nanobodies and its effective application. Conclusion. Nanobodies can be used in many fields of medicine and biotechnology such as research, diagnostics and therapy of oncology, infectious, hematological, inflammatory, autoimmune and neurological diseases. They can also be easily modified using another nanobody, molecules or radioactive mark as necessary. Nanobodies have huge potential for applications in diagnostics, therapy and medicine.

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Woods, James. "Selection of Functional Intracellular Nanobodies." SLAS DISCOVERY: Advancing the Science of Drug Discovery 24, no. 7 (June 7, 2019): 703–13. http://dx.doi.org/10.1177/2472555219853235.

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Camelid-derived nanobodies are versatile tools for research, diagnostics, and therapeutics. Certain nanobodies can function as intrabodies and bind antigens within the eukaryotic cytosol. This capability is valuable for the development of intracellular probes and targeted gene therapies. Consequently, many attempts have been made to produce nanobodies that are intracellularly stable and resistant to aggregation. Pursuit of these intrabodies generally focuses on library design or nanobody selection method. Recent variations of library design have yielded diverse libraries capable of producing nanobodies against a wide variety of antigens. Novel screening methods have also been developed, yielding nanobodies with high affinity for intracellular antigens. These screening techniques can have advantages over phage display methods when nanobodies against intracellular antigens must be rapidly produced. Some intracellular screening methods convey the additional advantage of selecting for other desired intrabody characteristics, such as antiviral action or conditional stability. This review summarizes the recent developments in both library design and selection methods aimed at producing intrabodies.

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Liang, Liu, Zixi Hu, Yingying Huang, Siliang Duan, Jian He, Yong Huang, Yongxiang Zhao, and Xiaoling Lu. "Advances in Nanobodies." Journal of Nanoscience and Nanotechnology 16, no. 12 (December 1, 2016): 12099–111. http://dx.doi.org/10.1166/jnn.2016.13767.

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Strack, Rita. "Nanobodies made versatile." Nature Methods 20, no. 1 (January 2023): 37. http://dx.doi.org/10.1038/s41592-022-01757-z.

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Silva-Pilipich, Noelia, Cristian Smerdou, and Lucía Vanrell. "A Small Virus to Deliver Small Antibodies: New Targeted Therapies Based on AAV Delivery of Nanobodies." Microorganisms 9, no. 9 (September 15, 2021): 1956. http://dx.doi.org/10.3390/microorganisms9091956.

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Nanobodies are camelid-derived single-domain antibodies that present some advantages versus conventional antibodies, such as a smaller size, and higher tissue penetrability, stability, and hydrophilicity. Although nanobodies can be delivered as proteins, in vivo expression from adeno-associated viral (AAV) vectors represents an attractive strategy. This is due to the fact that AAV vectors, that can provide long-term expression of recombinant genes, have shown an excellent safety profile, and can accommodate genes for one or several nanobodies. In fact, several studies showed that AAV vectors can provide sustained nanobody expression both locally or systemically in preclinical models of human diseases. Some of the pathologies addressed with this technology include cancer, neurological, cardiovascular, infectious, and genetic diseases. Depending on the indication, AAV-delivered nanobodies can be expressed extracellularly or inside cells. Intracellular nanobodies or “intrabodies” carry out their function by interacting with cell proteins involved in disease and have also been designed to help elucidate cellular mechanisms by interfering with normal cell processes. Finally, nanobodies can also be used to retarget AAV vectors, when tethered to viral capsid proteins. This review covers applications in which AAV vectors have been used to deliver nanobodies, with a focus on their therapeutic use.

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Adel M, Zakri, AL-Doss Abdullah A, Sack Markus, Ali Ahmed A, Samara Emad M, Ahmed Basem S, Amer Mahmoud A, Abdalla Omar A, and Al-Saleh Mohammed A. "Cloning and characterisation of nanobodies against the coat protein of Zucchini yellow mosaic virus." Plant Protection Science 54, No. 4 (August 25, 2018): 215–21. http://dx.doi.org/10.17221/158/2017-pps.

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Zucchini yellow mosaic virus (ZYMV), in the family Potyviridae, causes an economically important disease. Antibodies are valuable reagents for diagnostic assays to rapidly detect viral infection. Here, we report the isolation of camel-derived variable domains of the heavy chain antibody (VHH, also called nanobodies) directed against the coat protein (CP) of ZYMV. Several nanobodies that specifically recognise ZYMV-CP were identified. The isolated nanobodies showed binding not only to recombinant ZYMV-CP but also to native ZYMV, indicating that these nanobodies can be used in diagnostic tools to detect viral infections.

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Deszyński, Piotr, Jakub Młokosiewicz, Adam Volanakis, Igor Jaszczyszyn, Natalie Castellana, Stefano Bonissone, Rajkumar Ganesan, and Konrad Krawczyk. "INDI—integrated nanobody database for immunoinformatics." Nucleic Acids Research 50, no. D1 (November 8, 2021): D1273—D1281. http://dx.doi.org/10.1093/nar/gkab1021.

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Abstract Nanobodies, a subclass of antibodies found in camelids, are versatile molecular binding scaffolds composed of a single polypeptide chain. The small size of nanobodies bestows multiple therapeutic advantages (stability, tumor penetration) with the first therapeutic approval in 2018 cementing the clinical viability of this format. Structured data and sequence information of nanobodies will enable the accelerated clinical development of nanobody-based therapeutics. Though the nanobody sequence and structure data are deposited in the public domain at an accelerating pace, the heterogeneity of sources and lack of standardization hampers reliable harvesting of nanobody information. We address this issue by creating the Integrated Database of Nanobodies for Immunoinformatics (INDI, http://naturalantibody.com/nanobodies). INDI collates nanobodies from all the major public outlets of biological sequences: patents, GenBank, next-generation sequencing repositories, structures and scientific publications. We equip INDI with powerful nanobody-specific sequence and text search facilitating access to >11 million nanobody sequences. INDI should facilitate development of novel nanobody-specific computational protocols helping to deliver on the therapeutic promise of this drug format.

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Zhang, Caixia, Weiqi Zhang, Xiaoqian Tang, Qi Zhang, Wen Zhang, and Peiwu Li. "Change of Amino Acid Residues in Idiotypic Nanobodies Enhanced the Sensitivity of Competitive Enzyme Immunoassay for Mycotoxin Ochratoxin A in Cereals." Toxins 12, no. 4 (April 23, 2020): 273. http://dx.doi.org/10.3390/toxins12040273.

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Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody’s primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I–L), two of them in CDR2 (G–D, E–K), and three of them in CDR3 (Y–H, Y–W). Second, the affinity constant of NCA1 was tested as 1.20 × 108 L mol−1, which is about 4 times lower than that of NCA2 (5.36 × 108 L mol−1). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL−1, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL−1). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL−1 with a limit of detection (LOD) of 0.003 ng mL−1. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1–12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals.

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Kumar, Meenakshi Sundaram, Megan E. Fowler-Magaw, Daniel Kulick, Sivakumar Boopathy, Del Hayden Gadd, Melissa Rotunno, Catherine Douthwright, et al. "Anti-SOD1 Nanobodies That Stabilize Misfolded SOD1 Proteins Also Promote Neurite Outgrowth in Mutant SOD1 Human Neurons." International Journal of Molecular Sciences 23, no. 24 (December 16, 2022): 16013. http://dx.doi.org/10.3390/ijms232416013.

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ALS-linked mutations induce aberrant conformations within the SOD1 protein that are thought to underlie the pathogenic mechanism of SOD1-mediated ALS. Although clinical trials are underway for gene silencing of SOD1, these approaches reduce both wild-type and mutated forms of SOD1. Here, we sought to develop anti-SOD1 nanobodies with selectivity for mutant and misfolded forms of human SOD1 over wild-type SOD1. Characterization of two anti-SOD1 nanobodies revealed that these biologics stabilize mutant SOD1 in vitro. Further, SOD1 expression levels were enhanced and the physiological subcellular localization of mutant SOD1 was restored upon co-expression of anti-SOD1 nanobodies in immortalized cells. In human motor neurons harboring the SOD1 A4V mutation, anti-SOD1 nanobody expression promoted neurite outgrowth, demonstrating a protective effect of anti-SOD1 nanobodies in otherwise unhealthy cells. In vitro assays revealed that an anti-SOD1 nanobody exhibited selectivity for human mutant SOD1 over endogenous murine SOD1, thus supporting the preclinical utility of anti-SOD1 nanobodies for testing in animal models of ALS. In sum, the anti-SOD1 nanobodies developed and presented herein represent viable biologics for further preclinical testing in human and mouse models of ALS.

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Mukhametova, Lilia I., Sergei A. Eremin, Dmitrii A. Arutyunyan, Oksana S. Goryainova, Tatiana I. Ivanova, and Sergei V. Tillib. "Fluorescence Polarization Immunoassay of Human Lactoferrin in Milk Using Small Single-Domain Antibodies." Biochemistry (Moscow) 87, no. 12-13 (December 2022): 1679–88. http://dx.doi.org/10.1134/s0006297922120227.

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Abstract Due to its unique structure and properties, human breast milk lactoferrin (hLF) has many nutritional and health-promoting functions in infants, including protection against inflammation and bacterial infections. The lack of LF in breastmilk or formula can result in the weakening of the infant’s immune system. Noncompetitive polarization fluorescence immunoassay (FPIA) is a promising method for hLF quantification in milk and dairy products, which does not require the separation of the bound and free protein and allows to avoid time-consuming sample preparation. The use of fluorescently labeled single-domain camelid antibodies (nanobodies) for protein recognition in FPIA makes it possible to quantify relatively large antigens, in particular, hLF. In this work, we used previously obtained fluorescein isothiocyanate (FITC)-conjugated anti-hLF5 and anti-hLF16 nanobodies, which selectively recognized two different human lactoferrin epitopes, but did not bind to goat lactoferrin. The kinetics of hLF interaction with the FITC-labeled nanobodies was studied. The dissociation constant (KD) for the anti-LF5 and anti-LF16 nanobodies was 3.2 ± 0.3 and 4.9 ± 0.4 nM, respectively, indicating the high-affinity binding of these nanobodies to hLF. We developed the FPIA protocol and determined the concentration of FITC-labeled anti-hLF5 and anti-hLF16 nanobodies that provided the optimal fluorescence signal and stable fluorescence polarization value. We also studied the dependence of fluorescence polarization on the hLF concentration in the noncompetitive FPIA with FITC-anti-hLF5 nanobody. The detection limit for hLF was 2.1 ± 0.2 µg/ml and the linear range for determining the hLF concentration was 3-10 µg/ml. FPIA is commonly used to assay low-molecular-weight substances; however, the use of fluorescently labeled nanobodies allows quantification of high-molecular-weight proteins. Here, we demonstrated that FPIA with fluorescently labeled nanobodies can be used for hLF quantification in milk.

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Tillib, Sergei V., Oksana S. Goryainova, Anastasiya M. Sachko, and Tatiana I. Ivanova. "High-Affinity Single-Domain Antibodies for Analyzing Human Apo- and Holo-Transferrin." Acta Naturae 14, no. 2 (July 21, 2022): 98–102. http://dx.doi.org/10.32607/actanaturae.11663.

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A highly efficient technology for generating new monoclonal single-domain recombinant antibodies (nanobodies) was used to obtain a panel of nanobodies recognizing human apo- and/or holo-transferrin. This article is devoted to the primary analysis of the properties of two different variants of the new nanobodies obtained by us, as well as to the demonstration of the unique potential of their application for diagnostic studies. The simultaneous use of immunosorbents based on these nanobodies apparently makes it possible to detect changes in the relative abundance of apo- and holo-transferrin in human biological fluids. Such changes could potentially be indicative of an increased risk or degree of development of pathological processes, such as malignant neoplasms in humans.

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Mashayekhi, Vida, Katerina T. Xenaki, Paul M. P. van Bergen en Henegouwen, and Sabrina Oliveira. "Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy." Cancers 12, no. 10 (September 23, 2020): 2732. http://dx.doi.org/10.3390/cancers12102732.

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Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer, although sub-optimal tumor specificity and side effects have hindered its clinical application. We introduced a new strategy named nanobody-targeted PDT in which photosensitizers are delivered to tumor cells by means of nanobodies. As efficacy of targeted PDT can be hampered by heterogeneity of target expression and/or moderate/low target expression levels, we explored the possibility of combined targeting of endothelial and cancer cells in vitro. We developed nanobodies binding to the mouse VEGFR2, which is overexpressed on tumor vasculature, and combined these with nanobodies specific for the cancer cell target EGFR. The nanobodies were conjugated to the photosensitizer IRDye700DX and specificity of the newly developed nanobodies was verified using several endothelial cell lines. The cytotoxicity of these conjugates was assessed in monocultures and in co-cultures with cancer cells, after illumination with an appropriate laser. The results show that the anti-VEGFR2 conjugates are specific and potent PDT agents. Nanobody-targeted PDT on co-culture of endothelial and cancer cells showed improved efficacy, when VEGFR2 and EGFR targeting nanobodies were applied simultaneously. Altogether, dual targeting of endothelial and cancer cells is a promising novel therapeutic strategy for more effective nanobody-targeted PDT.

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Liu, Bingying, and Daiwen Yang. "Easily Established and Multifunctional Synthetic Nanobody Libraries as Research Tools." International Journal of Molecular Sciences 23, no. 3 (January 27, 2022): 1482. http://dx.doi.org/10.3390/ijms23031482.

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Nanobodies, or VHHs, refer to the antigen-binding domain of heavy-chain antibodies (HCAbs) from camelids. They have been widely used as research tools for protein purification and structure determination due to their small size, high specificity, and high stability, overcoming limitations with conventional antibody fragments. However, animal immunization and subsequent retrieval of antigen-specific nanobodies are expensive and complicated. Construction of synthetic nanobody libraries using DNA oligonucleotides is a cost-effective alternative for immunization libraries and shows great potential in identifying antigen-specific or even conformation-specific nanobodies. This review summarizes and analyses synthetic nanobody libraries in the current literature, including library design and biopanning methods, and further discusses applications of antigen-specific nanobodies obtained from synthetic libraries to research.

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Zhu, Jingjing, Jeroen Declercq, Bart Roucourt, Gholamreza H. Ghassabeh, Sandra Meulemans, Jörg Kinne, Guido David, et al. "Generation and characterization of non-competitive furin-inhibiting nanobodies." Biochemical Journal 448, no. 1 (October 18, 2012): 73–82. http://dx.doi.org/10.1042/bj20120537.

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The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFβ (transforming growth factor β) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.

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Kang, Wei, Chuanfeng Ding, Danni Zheng, Xiao Ma, Lun Yi, Xinyi Tong, Chuang Wu, Chuang Xue, Yongsheng Yu, and Qian Zhou. "Nanobody Conjugates for Targeted Cancer Therapy and Imaging." Technology in Cancer Research & Treatment 20 (January 1, 2021): 153303382110101. http://dx.doi.org/10.1177/15330338211010117.

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Conventional antibody-based targeted cancer therapy is one of the most promising avenues of successful cancer treatment, with the potential to reduce toxic side effects to healthy cells surrounding tumor cells. However, the full potential of antibodies is severely limited due to their large size, low stability, slow clearance, and high immunogenicity. Alternatively, recently discovered nanobodies, which are the smallest naturally occurring antigen-binding format, have shown great potential for addressing these limitations. Bioconjugation of nanobodies to functional groups such as toxins, enzymes, radionucleotides, and fluorophores can improve the efficacy and potency of nanobodies, enhance their in vivo pharmacokinetics, and expand the range of potential applications. Herein, we review the superior characteristics of nanobodies in comparison to conventional antibodies and provide insight into recent developments in nanobody conjugates for targeted cancer therapy and imaging.

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Matamoros, Alcivar, Esther, Ivanova,, and Maily, Selena, González, Avilés. "Study review of camelid and shark antibodies for biomedical and biotechnological applications." Bionatura 6, no. 4 (November 15, 2021): 2331–40. http://dx.doi.org/10.21931/rb/2021.06.04.31.

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The antibodies of camelids and sharks are about one–half of the conventional ones while regular antibodies have four protein chains: two light and two heavy, these small antibodies studied have just two heavy chains; they lack a light chain. In recent years, nanobodies have been the focus of attention because they can recognize epitopes that are usually not antigenic (hidden) for conventional antibodies. On the clinical side, researchers are testing nanobodies (Nbs) in the fight against diseases and disease diagnosis. Nanobodies also are attractive because they can prevent protein aggregation and clear the already existing aggregates. Furthermore, new treatments using these Nbs can neutralize the severe acute respiratory syndrome coronavirus (SARS-CoV-2) for preventing COVID-19. In this review, we sum up recent findings of the proposed nanobodies for their potential application.

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Klein, A., S. Hank, A. Raulf, E. F. Joest, F. Tissen, M. Heilemann, R. Wieneke, and R. Tampé. "Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies." Chemical Science 9, no. 40 (2018): 7835–42. http://dx.doi.org/10.1039/c8sc02910e.

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Using engineered nanobodies with bright organic dyes (fluorescent nanobodies) and subsequent microfluidic cell manipulation, controlled nanobody delivery was achieved, allowing the multiplexed imaging and super-resolution of endogenous protein networks in living cells.

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Farrants, Helen, Miroslaw Tarnawski, Thorsten G. Müller, Shotaro Otsuka, Julien Hiblot, Birgit Koch, Moritz Kueblbeck, Hans-Georg Kräusslich, Jan Ellenberg, and Kai Johnsson. "Chemogenetic Control of Nanobodies." Nature Methods 17, no. 3 (February 17, 2020): 279–82. http://dx.doi.org/10.1038/s41592-020-0746-7.

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Ingram, Jessica R., Florian I. Schmidt, and Hidde L. Ploegh. "Exploiting Nanobodies’ Singular Traits." Annual Review of Immunology 36, no. 1 (April 26, 2018): 695–715. http://dx.doi.org/10.1146/annurev-immunol-042617-053327.

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Nicholls, Henry. "Nanobodies: The ultrasmall antibodies." New Scientist 196, no. 2624 (October 2007): 50–53. http://dx.doi.org/10.1016/s0262-4079(07)62536-6.

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Vaneycken, Ilse, Matthias D’huyvetter, Sophie Hernot, Jens De Vos, Catarina Xavier, Nick Devoogdt, Vicky Caveliers, and Tony Lahoutte. "Immuno-imaging using nanobodies." Current Opinion in Biotechnology 22, no. 6 (December 2011): 877–81. http://dx.doi.org/10.1016/j.copbio.2011.06.009.

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Majerle, Andreja, San Hadži, Jana Aupič, Tadej Satler, Fabio Lapenta, Žiga Strmšek, Jurij Lah, Remy Loris, and Roman Jerala. "A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures." Proceedings of the National Academy of Sciences 118, no. 17 (April 23, 2021): e2021899118. http://dx.doi.org/10.1073/pnas.2021899118.

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Coiled-coil (CC) dimers are widely used in protein design because of their modularity and well-understood sequence–structure relationship. In CC protein origami design, a polypeptide chain is assembled from a defined sequence of CC building segments that determine the self-assembly of protein cages into polyhedral shapes, such as the tetrahedron, triangular prism, or four-sided pyramid. However, a targeted functionalization of the CC modules could significantly expand the versatility of protein origami scaffolds. Here, we describe a panel of single-chain camelid antibodies (nanobodies) directed against different CC modules of a de novo designed protein origami tetrahedron. We show that these nanobodies are able to recognize the same CC modules in different polyhedral contexts, such as isolated CC dimers, tetrahedra, triangular prisms, or trigonal bipyramids, thereby extending the ability to functionalize polyhedra with nanobodies in a desired stoichiometry. Crystal structures of five nanobody-CC complexes in combination with small-angle X-ray scattering show binding interactions between nanobodies and CC dimers forming the edges of a tetrahedron with the nanobody entering the tetrahedral cavity. Furthermore, we identified a pair of allosteric nanobodies in which the binding to the distant epitopes on the antiparallel homodimeric APH CC is coupled via a strong positive cooperativity. A toolbox of well-characterized nanobodies specific for CC modules provides a unique tool to target defined sites in the designed protein structures, thus opening numerous opportunities for the functionalization of CC protein origami polyhedra or CC-based bionanomaterials.

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Niu, Zhiyuan, Zhixia Luo, Pengyang Sun, Linwei Ning, Xinru Jin, Guanxu Chen, Changjiang Guo, Lingtong Zhi, Wei Chang, and Wuling Zhu. "In Vitro Nanobody Library Construction by Using Gene Designated-Region Pan-Editing Technology." BioDesign Research 2022 (August 2, 2022): 1–9. http://dx.doi.org/10.34133/2022/9823578.

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Camelid single-domain antibody fragments (nanobodies) are an emerging force in therapeutic biopharmaceuticals and clinical diagnostic reagents in recent years. Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting the large-scale application of nanobodies. In this study, we developed three kinds of gene designated-region pan-editing (GDP) technologies to introduce multiple mutations in complementarity-determining regions (CDRs) of nanobodies in vitro. Including the integration of G-quadruplex fragments in CDRs, which induces the spontaneous multiple mutations in CDRs; however, these mutant sequences are highly similar, resulting in a lack of sequences diversity in the CDRs. We also used CDR-targeting traditional gRNA-guided base-editors, which effectively diversify the CDRs. And most importantly, we developed the self-assembling gRNAs, which are generated by reprogrammed tracrRNA hijacking of endogenous mRNAs as crRNAs. Using base-editors guided by self-assembling gRNAs, we can realize the iteratively diversify the CDRs. And we believe the last GDP technology is highly promising in immunization-free nanobody library construction, and the full development of this novel nanobody discovery platform can realize the synthetic evolution of nanobodies in vitro.

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Naidoo, Dhaneshree Bestinee, and Anil Amichund Chuturgoon. "Nanobodies Enhancing Cancer Visualization, Diagnosis and Therapeutics." International Journal of Molecular Sciences 22, no. 18 (September 10, 2021): 9778. http://dx.doi.org/10.3390/ijms22189778.

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Worldwide, cancer is a serious health concern due to the increasing rates of incidence and mortality. Conventional cancer imaging, diagnosis and treatment practices continue to substantially contribute to the fight against cancer. However, these practices do have some risks, adverse effects and limitations, which can affect patient outcomes. Although antibodies have been developed, successfully used and proven beneficial in various oncology practices, the use of antibodies also comes with certain challenges and limitations (large in size, poor tumor penetration, high immunogenicity and a long half-life). Therefore, it is vital to develop new ways to visualize, diagnose and treat cancer. Nanobodies are novel antigen-binding fragments that possess many advantageous properties (small in size, low immunogenicity and a short half-life). Thus, the use of nanobodies in cancer practices may overcome the challenges experienced with using traditional antibodies. In this review, we discuss (1) the challenges with antibody usage and the superior qualities of nanobodies; (2) the use of antibodies and nanobodies in cancer imaging, diagnosis, drug delivery and therapy (surgery, radiotherapy, chemotherapy and immunotherapy); and (3) the potential improvements in oncology practices due to the use of nanobodies as compared to antibodies.

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Küppers, Jim, Stefan Kürpig, Ralph A. Bundschuh, Markus Essler, and Susanne Lütje. "Radiolabeling Strategies of Nanobodies for Imaging Applications." Diagnostics 11, no. 9 (August 25, 2021): 1530. http://dx.doi.org/10.3390/diagnostics11091530.

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Nanobodies are small recombinant antigen-binding fragments derived from camelid heavy-chain only antibodies. Due to their compact structure, pharmacokinetics of nanobodies are favorable compared to full-size antibodies, allowing rapid accumulation to their targets after intravenous administration, while unbound molecules are quickly cleared from the circulation. In consequence, high signal-to-background ratios can be achieved, rendering radiolabeled nanobodies high-potential candidates for imaging applications in oncology, immunology and specific diseases, for instance in the cardiovascular system. In this review, a comprehensive overview of central aspects of nanobody functionalization and radiolabeling strategies is provided.

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Valdés-Tresanco, Mario S., Andrea Molina-Zapata, Alaín González Pose, and Ernesto Moreno. "Structural Insights into the Design of Synthetic Nanobody Libraries." Molecules 27, no. 7 (March 28, 2022): 2198. http://dx.doi.org/10.3390/molecules27072198.

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Single domain antibodies from camelids, or nanobodies, are a unique class of antibody fragments with several advantageous characteristics: small monomeric size, high stability and solubility and easy tailoring for multiple applications. Nanobodies are gaining increasing acceptance as diagnostic tools and promising therapeutic agents in cancer and other diseases. While most nanobodies are obtained from immunized animals of the camelid family, a few synthetic nanobody libraries constructed in recent years have shown the capability of generating high quality nanobodies in terms of affinity and stability. Since this synthetic approach has important advantages over the use of animals, the recent advances are indeed encouraging. Here we review over a dozen synthetic nanobody libraries reported so far and discuss the different approaches followed in their construction and validation, with an emphasis on framework and hypervariable loop design as critical issues defining their potential as high-class nanobody sources.

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Lecocq, Quentin, Katty Zeven, Yannick De Vlaeminck, Sandrina Martens, Sam Massa, Cleo Goyvaerts, Geert Raes, Marleen Keyaerts, Karine Breckpot, and Nick Devoogdt. "Noninvasive Imaging of the Immune Checkpoint LAG-3 Using Nanobodies, from Development to Pre-Clinical Use." Biomolecules 9, no. 10 (September 29, 2019): 548. http://dx.doi.org/10.3390/biom9100548.

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Immune checkpoint inhibition (ICI) is a promising cancer therapy, which has progressed rapidly from a preclinical concept to clinical implementation. Commonly considered targets in ICI are CTLA-4, PD-1/PD-L1, and LAG-3, and the list grows. As ICI is generally only beneficial for a subset of patients, there is a need to select patients that are eligible for therapy as well as to monitor therapy response. There is growing interest to do this noninvasively, by molecular imaging with target-specific tracers. To this day, noninvasive imaging has focused on CTLA-4 and PD-1/PD-L1, while there is no noninvasive tool available to accurately assess LAG-3 expression in vivo. In this proof-of-concept study, we developed nanobodies, the smallest functional fragments from camelid heavy chain-only antibodies, to noninvasively evaluate mouse LAG-3 expression using single positron emission computed tomography (SPECT)/CT imaging. The in vitro characterization of 114 nanobodies led to the selection of nine nanobodies binding to mouse LAG-3. The injection of 99mTechnetium-labeled nanobodies in healthy mice showed specific uptake in immune peripheral organs like the spleen and lymph nodes, which was not observed in LAG-3 gene knock-out mice. Moreover, nanobody uptake could be visualized using SPECT/CT and correlated to the presence of LAG-3 as assessed in flow cytometry and immunohistochemistry. SPECT/CT scans of tumor bearing mice further confirmed the diagnostic potential of the nanobodies. These findings substantiate the approach to use nanobodies as a tool to image inhibitory immune checkpoints in the tumor environment.

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Abderrazek, Rahma Ben, Issam Hmila, Cécile Vincke, Zakaria Benlasfar, Mireille Pellis, Hafedh Dabbek, Dirk Saerens, Mohamed El Ayeb, Serge Muyldermans, and Balkiss Bouhaouala-Zahar. "Identification of potent nanobodies to neutralize the most poisonous polypeptide from scorpion venom." Biochemical Journal 424, no. 2 (November 11, 2009): 263–72. http://dx.doi.org/10.1042/bj20090697.

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Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patient, causes serious medical problems. Nanobodies, single-domain antigen-binding fragments derived from dromedary heavy-chain antibodies, have a size that closely matches that of scorpion toxins. Therefore these nanobodies might be developed into potent immunotherapeutics to treat scorpion envenoming. Multiple nanobodies of sub-nanomolar affinity to AahII, the most toxic polypeptide within the Androctonus australis hector venom, were isolated from a dromedary immunized with AahII. These nanobodies neutralize the lethal effect of AahII to various extents without clear correlation with the kinetic rate constants kon or koff, or the equilibrium dissociation constant, KD. One particular nanobody, referred to as NbAahII10, which targets a unique epitope on AahII, neutralizes 7 LD50 of this toxin in mice, corresponding to a neutralizing capacity of approx. 37000 LD50 of AahII/mg of nanobody. Such high neutralizing potency has never been reached before by any other monoclonal antibody fragment.

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Verkhivker, Gennady. "Structural and Computational Studies of the SARS-CoV-2 Spike Protein Binding Mechanisms with Nanobodies: From Structure and Dynamics to Avidity-Driven Nanobody Engineering." International Journal of Molecular Sciences 23, no. 6 (March 8, 2022): 2928. http://dx.doi.org/10.3390/ijms23062928.

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Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.

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Zottel, Alja, Ivana Jovčevska, Neja Šamec, Jernej Mlakar, Jernej Šribar, Igor Križaj, Marija Skoblar Vidmar, and Radovan Komel. "Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration." Therapeutic Advances in Medical Oncology 12 (January 2020): 175883592091530. http://dx.doi.org/10.1177/1758835920915302.

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Background: Glioblastoma is a particularly common and very aggressive primary brain tumour. One of the main causes of therapy failure is the presence of glioblastoma stem cells that are resistant to chemotherapy and radiotherapy, and that have the potential to form new tumours. This study focuses on validation of eight novel antigens, TRIM28, nucleolin, vimentin, nucleosome assembly protein 1-like 1 (NAP1L1), mitochondrial translation elongation factor (EF-TU) (TUFM), dihydropyrimidinase-related protein 2 (DPYSL2), collapsin response mediator protein 1 (CRMP1) and Aly/REF export factor (ALYREF), as putative glioblastoma targets, using nanobodies. Methods: Expression of these eight antigens was analysed at the cellular level by qPCR, ELISA and immunocytochemistry, and in tissues by immunohistochemistry. The cytotoxic effects of the nanobodies were determined using AlamarBlue and water-soluble tetrazolium tests. Annexin V/propidium iodide tests were used to determine apoptotsis/necrosis of the cells in the presence of the nanobodies. Cell migration assays were performed to determine the effects of the nanobodies on cell migration. Results: NAP1L1 and CRMP1 were significantly overexpressed in glioblastoma stem cells in comparison with astrocytes and glioblastoma cell lines at the mRNA and protein levels. Vimentin, DPYSL2 and ALYREF were overexpressed in glioblastoma cell lines only at the protein level. The functional part of the study examined the cytotoxic effects of the nanobodies on glioblastoma cell lines. Four of the nanobodies were selected in terms of their specificity towards glioblastoma cells and protein overexpression: anti-vimentin (Nb79), anti-NAP1L1 (Nb179), anti-TUFM (Nb225) and anti-DPYSL2 (Nb314). In further experiments to optimise the nanobody treatment schemes, to increase their effects, and to determine their impact on migration of glioblastoma cells, the anti-TUFM nanobody showed large cytotoxic effects on glioblastoma stem cells, while the anti-vimentin, anti-NAP1L1 and anti-DPYSL2 nanobodies were indicated as agents to target mature glioblastoma cells. The anti-vimentin nanobody also had significant effects on migration of mature glioblastoma cells. Conclusion: Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) nanobodies are indicated for further examination for cell targeting. The anti-TUFM nanobody, Nb225, is particularly potent for inhibition of cell growth after long-term exposure of glioblastoma stem cells, with minor effects seen for astrocytes. The anti-vimentin nanobody represents an agent for inhibition of cell migration.

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Gormal, Rachel S., Pranesh Padmanabhan, Ravikiran Kasula, Adekunle T. Bademosi, Sean Coakley, Jean Giacomotto, Ailisa Blum, et al. "Modular transient nanoclustering of activated β2-adrenergic receptors revealed by single-molecule tracking of conformation-specific nanobodies." Proceedings of the National Academy of Sciences 117, no. 48 (November 19, 2020): 30476–87. http://dx.doi.org/10.1073/pnas.2007443117.

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None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous β2-adrenoreceptor (β2-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated β2-AR(Nb80) is highly immobile and organized in nanoclusters. The Gαs−GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated β2-AR(Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.

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Smith, Caroline Noel, Kyle Kihn, Zachary A. Williamson, K. Martin Chow, Louis B. Hersh, Konstantin Korotkov, Daniel Deredge, and Jessica S. Blackburn. "Abstract 672: Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3)." Cancer Research 82, no. 12_Supplement (June 15, 2022): 672. http://dx.doi.org/10.1158/1538-7445.am2022-672.

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Abstract Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual-specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and is correlated with poor patient prognosis in a variety of solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. The development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is shallow and hydrophobic. Currently available small molecules do not exhibit binding specificity for PRL-3 over PRL family members, and the only research antibody specific for PRL-3 can only recognize denatured protein. To address the lack of tools available to study PRL-3, we have developed alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3. Nanobodies have emerged as a valuable research tool and show promise as cancer therapeutics as they are ~15kD and lack light chains, allowing them to reach cavities within active sites that conventional antibodies cannot normally reach. Nanobodies also maintain high specificity and affinity for their antigens. We identified seven unique nanobodies that bind to PRL-3 with no activity towards PRL-1 and PRL-2, making our nanobodies one of the first tools to selectively target PRL-3 in its native state. We used biolayer interferometry and found the nanobody binding affinity for PRL-3 to be within a KD of 30 - 300 nM, similar to that of antibodies currently on the market. We identified PRL-3:nanobody interactions with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and showed binding outside the active site. These data were confirmed by analyzing the effects of nanobodies on PRL-3 phosphatase activity and substrate binding. Our anti-PRL-3 nanobodies specifically pulled down PRL-3 over PRL-1/-2 in immunoprecipitation experiments. Finally, we used these nanobodies to analyze PRL-3 localization in fixed immunofluorescence experiments in human cancer cells. We found that a C-terminal tag on PRL-3, such as FLAG or GFP, enhanced PRL-3 localization to the membrane, compared to untagged protein, which may have confounded previous PRL-3 functional studies. We are currently utilizing these nanobodies in two ways to understand PRL-3’s role in cancer. First, we will use the nanobody to stabilize PRL-3 for X-ray crystallography to develop higher resolution structures that could contribute to substrate identification and drug design. Secondly, we will examine PRL-3 function and trafficking during various cancer processes, such as proliferation, invasion, and stress, to determine how PRL-3 localization contributes to cancer progression. Citation Format: Caroline Noel Smith, Kyle Kihn, Zachary A. Williamson, K. Martin Chow, Louis B. Hersh, Konstantin Korotkov, Daniel Deredge, Jessica S. Blackburn. Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 672.

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Duhoo, Yoan, Jennifer Roche, Thi Trang Nhung Trinh, Aline Desmyter, Anaïs Gaubert, Christine Kellenberger, Christian Cambillau, Alain Roussel, and Philippe Leone. "Camelid nanobodies used as crystallization chaperones for different constructs of PorM, a component of the type IX secretion system fromPorphyromonas gingivalis." Acta Crystallographica Section F Structural Biology Communications 73, no. 5 (April 26, 2017): 286–93. http://dx.doi.org/10.1107/s2053230x17005969.

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PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) inPorphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.

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Zhou, Xiaohua, Maarten L. V. Hendrickx, Gholamreza Hassanzadeh-Ghassabeh, Serge Muyldermans, and Paul J. Declerck. "Generation and in vitro characterisation of inhibitory nanobodies towards plasminogen activator inhibitor 1." Thrombosis and Haemostasis 116, no. 12 (November 2016): 1032–40. http://dx.doi.org/10.1160/th16-04-0306.

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SummaryPlasminogen activator inhibitor 1 (PAI-1) is the principal physiological inhibitor of tissue-type plasminogen activator (t-PA) and has been identified as a risk factor in cardiovascular diseases. In order to generate nanobodies against PAI-1 to interfere with its functional properties, we constructed three nanobody libraries upon immunisation of three alpacas with three different PAI-1 variants. Three panels of nanobodies were selected against these PAI-1 variants. Evaluation of the amino acid sequence identity of the complementarity determining region-3 (CDR3) reveals 34 clusters in total. Five nanobodies (VHH-s-a98, VHH-2w-64, VHH-s-a27, VHH-s-a93 and VHH-2g-42) representing five clusters exhibit inhibition towards PAI-1 activity. VHH-s-a98 and VHH-2w-64 inhibit both glycosylated and non-glycosylated PAI-1 variants through a substrate-inducing mechanism, and bind to two different regions close to αhC and the hinge region of αhF; the profibrinolytic effect of both nanobodies was confirmed using an in vitro clot lysis assay. VHH-s-a93 may inhibit PAI-1 activity by preventing the formation of the initial PAI-1•t-PA complex formation and binds to the hinge region of the reactive centre loop. Epitopes of VHH-s-a27 and VHH-2g-42 could not be deduced yet. These five nanobodies interfere with PAI-1 activity through different mechanisms and merit further evaluation for the development of future profibrinolytic therapeutics.

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Hassanzadeh-Ghassabeh, Gholamreza, Nick Devoogdt, Pieter De Pauw, Cécile Vincke, and Serge Muyldermans. "Nanobodies and their potential applications." Nanomedicine 8, no. 6 (June 2013): 1013–26. http://dx.doi.org/10.2217/nnm.13.86.

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Barakat, Sarah, Melike Berksöz, Pegah Zahedimaram, Sofia Piepoli, and Batu Erman. "Nanobodies as molecular imaging probes." Free Radical Biology and Medicine 182 (March 2022): 260–75. http://dx.doi.org/10.1016/j.freeradbiomed.2022.02.031.

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Muyldermans, Serge. "Nanobodies: Natural Single-Domain Antibodies." Annual Review of Biochemistry 82, no. 1 (June 2, 2013): 775–97. http://dx.doi.org/10.1146/annurev-biochem-063011-092449.

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Abderrazek, Rahma Ben, Issam Hmila, Cécile Vincke, Zakaria Benlasfar, Hafedh Dabbek, Mohamed El Ayeb, Serge Muyldermans, and Balkiss Bouhaouala-Zahar. "Nanobodies against AahII scorpion toxin." Toxicology Letters 189 (September 2009): S171. http://dx.doi.org/10.1016/j.toxlet.2009.06.701.

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Cheloha, Ross W., Thibault J. Harmand, Charlotte Wijne, Thomas U. Schwartz, and Hidde L. Ploegh. "Exploring cellular biochemistry with nanobodies." Journal of Biological Chemistry 295, no. 45 (August 31, 2020): 15307–27. http://dx.doi.org/10.1074/jbc.rev120.012960.

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Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain–only antibodies, discovered in camelids, have been truncated to yield single-domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.

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Martinez-Delgado, Gustavo. "Inhaled nanobodies against COVID-19." Nature Reviews Immunology 20, no. 10 (September 1, 2020): 593. http://dx.doi.org/10.1038/s41577-020-00443-5.

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Jovčevska, Ivana, and Serge Muyldermans. "The Therapeutic Potential of Nanobodies." BioDrugs 34, no. 1 (November 4, 2019): 11–26. http://dx.doi.org/10.1007/s40259-019-00392-z.

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Wagner, Hanna, Sarah Wehrle, Etienne Weiss, Marco Cavallari, and Wilfried Weber. "A Two-Step Approach for the Design and Generation of Nanobodies." International Journal of Molecular Sciences 19, no. 11 (November 2, 2018): 3444. http://dx.doi.org/10.3390/ijms19113444.

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Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols.

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Yamagata, Masahito, and Joshua R. Sanes. "Reporter–nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents." Proceedings of the National Academy of Sciences 115, no. 9 (February 13, 2018): 2126–31. http://dx.doi.org/10.1073/pnas.1722491115.

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Sensitive and specific antibodies are essential for detecting molecules in cells and tissues. However, currently used polyclonal and monoclonal antibodies are often less specific than desired, difficult to produce, and available in limited quantities. A promising recent approach to circumvent these limitations is to employ chemically defined antigen-combining domains called “nanobodies,” derived from single-chain camelid antibodies. Here, we used nanobodies to prepare sensitive unimolecular detection reagents by genetically fusing cDNAs encoding nanobodies to enzymatic or antigenic reporters. We call these fusions between a reporter and a nanobody “RANbodies.” They can be used to localize epitopes and to amplify signals from fluorescent proteins. They can be generated and purified simply and in unlimited amounts and can be preserved safely and inexpensively in the form of DNA or digital sequence.

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Güttler, Thomas, Matthias Dobbelstein, and Dirk Görlich. "Therapeutische Nanobodies gegen SARS-CoV-2." BIOspektrum 28, no. 1 (February 2022): 39–42. http://dx.doi.org/10.1007/s12268-022-1684-y.

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AbstractMonoclonal immunoglobulins are widely successful as therapeutics and have also been effective in treating COVID-19. However, their production in mammalian cells is expensive and cannot be scaled to meet the demand in a global pandemic. Camelid VHH antibodies (also called nanobodies), however, can be manufactured cost-efficiently in bacteria or yeast. Here we highlight our progress in developing nanobodies that effectively neutralize SARS-CoV-2 and its variants.

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Hanack, Katja, Anja Schlör, Pamela Holzlöhner, Martin Listek, Cindy Bauer, Monique Butze, Burkhard Micheel, et al. "Camelid nanobodies specific to human pancreatic glycoprotein 2." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 209.8. http://dx.doi.org/10.4049/jimmunol.196.supp.209.8.

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Abstract Pancreatic secretory zymogen-granule membrane glycoprotein 2 (GP2) has been identified to be a major autoantigenic target in Crohn’s disease patients. It was discussed recently that a long and a short isoform of GP2 exists whereas the short isoform is often detected by GP2-specific autoantibodies. In the outcome of inflammatory bowel diseases, these GP2-specific autoantibodies are discussed as new serological markers for diagnosis and therapeutic monitoring. To investigate this further, camelid nanobodies were generated by phage display and selected against the short isoform of GP2 in order to isolate specific tools for the discrimination of both isoforms. Nanobodies are single domain antibodies derived from camelid heavy chain only antibodies and characterized by a high stability and solubility. The selected candidates were expressed, purified and validated regarding their binding properties in different enzyme-linked immunosorbent assays formats, immunofluorescence, immunohistochemistry and surface plasmon resonance spectroscopy. Four different nanobodies could be selected whereof three recognize the short isoform of GP2 very specifically and one nanobody showed a high binding capacity for both isoforms. The KD values measured for all nanobodies were between 1.3 nM and 2.3 pM indicating highly specific binders suitable for the application as diagnostic tool in inflammatory bowel disease.

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Arce, Nicholas A., Ally J. Su, and Renhao Li. "Nanobody Activators of Von Willebrand Factor Via Targeting Its Autoinhibitory Module." Blood 138, Supplement 1 (November 5, 2021): 2074. http://dx.doi.org/10.1182/blood-2021-151052.

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Abstract Introduction: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein responsible for platelet arrest during injury, especially at high shear. After immobilization to the vessel wall, a VWF multimer is unfurled and elongated. This leads to exposure of the A1 domain therein that in turn binds to platelet receptor GPIbα and starts the aggregation process. Recently, it was suggested that VWF activation involves force-dependent disruption of the autoinhibitory module (AIM) that flanks the A1 domain on both sides. In this scenario, the AIM could be targeted for both VWF inhibition (Caplacizumab) and activation (ristocetin), although the exact mechanism and binding site of ristocetin still remains murky. If the quasi-stable structure of the AIM is important to VWF autoinhibition, specific disruption of its confirmation may be able to activate VWF. To this end, we sought to identify AIM-targeting activators using yeast surface display of a llama nanobody library. Methods: One adult Lama glama was immunized with recombinant human VWF AIM-A1 protein produced from transfected Expi293F cells. VHH specific genes were amplified from cDNAs prepared from PBMCs of the animal and electroporated into EBY100 cells. The resulting yeast display library was screened for AIM-specific binders via selection against binding to recombinant A1 protein without an intact AIM, and then for binding to the complex of AIM-A1 with GPIbα. Positive hits were produced as His-tagged monomeric nanobodies in E. coli and purified with nickel-affinity and gel filtration chromatography. The affinity of nanobodies to AIM-A1 was determined using bio-layer interferometry. Platelet-rich plasma from healthy donors was used to assess the effect of nanobodies on platelet aggregation in a light transmission aggregometer with comparison to that of ristocetin. Results: An AIM-A1-specific nanobody yeast display library was established. Several rounds of flow cytometry-based cell sorting of yeast cells with aforementioned binding properties produced AIM-binding nanobodies. Nanobodies encoded in three single clones have been expressed from E. coli and they exhibited differential binding affinities towards AIM-A1. Clone 6C4 showed the lowest affinity (K D 120 ± 3 nM), 6D12 showed intermediate affinity (K D 31 ± 0.8 nM), and 6C11 showed the highest affinity (K D 13.5 ± 0.2 nM) as shown in Figure 1. These nanobodies showed no detectable affinity towards recombinant A1-CAIM protein (residues 1268-1493), indicating that their epitopes are located in the N-terminal portion of the AIM (residues 1238-1267). When added to human platelet-rich plasma, each nanobody dose-dependently activated platelets and rapidly induced full platelet aggregation at concentrations exceeding the affinity of the nanobody for VWF (Figure 2). The aggregation could be inhibited by the addition of antibodies that block the interaction between VWF and GPIbα. Plots of extents of aggregation as a function of nanobody concentration produced EC 50 values of ~100 nM for 6C11 and 6D12. Conclusion: By isolating nanobodies that can bind specifically to the AIM and activate plasma VWF, we add supporting evidence that the AIM protects the A1 domain from binding to platelets. Interestingly, these nanobodies bind to the NAIM, on the opposite side of the module compared to ristocetin, the only known AIM-activating agent until now. With higher VWF-binding affinities than ristocetin and a robust profile as stable monomers, these nanobodies may prove useful in VWF-related research and diagnostics. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Verkhivker, Gennady. "Allosteric Determinants of the SARS-CoV-2 Spike Protein Binding with Nanobodies: Examining Mechanisms of Mutational Escape and Sensitivity of the Omicron Variant." International Journal of Molecular Sciences 23, no. 4 (February 16, 2022): 2172. http://dx.doi.org/10.3390/ijms23042172.

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Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against the SARS-CoV-2 virus and resilience against mutational escape. In this study, we performed a comprehensive computational analysis of the SARS-CoV-2 spike trimer complexes with single nanobodies Nb6, VHH E, and complex with VHH E/VHH V nanobody combination. We combined coarse-grained and all-atom molecular simulations and collective dynamics analysis with binding free energy scanning, perturbation-response scanning, and network centrality analysis to examine mechanisms of nanobody-induced allosteric modulation and cooperativity in the SARS-CoV-2 spike trimer complexes with these nanobodies. By quantifying energetic and allosteric determinants of the SARS-CoV-2 spike protein binding with nanobodies, we also examined nanobody-induced modulation of escaping mutations and the effect of the Omicron variant on nanobody binding. The mutational scanning analysis supported the notion that E484A mutation can have a significant detrimental effect on nanobody binding and result in Omicron-induced escape from nanobody neutralization. Our findings showed that SARS-CoV-2 spike protein might exploit the plasticity of specific allosteric hotspots to generate escape mutants that alter response to binding without compromising activity. The network analysis supported these findings showing that VHH E/VHH V nanobody binding can induce long-range couplings between the cryptic binding epitope and ACE2-binding site through a broader ensemble of communication paths that is less dependent on specific mediating centers and therefore may be less sensitive to mutational perturbations of functional residues. The results suggest that binding affinity and long-range communications of the SARS-CoV-2 complexes with nanobodies can be determined by structurally stable regulatory centers and conformationally adaptable hotspots that are allosterically coupled and collectively control resilience to mutational escape.

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Zarantonello, Alessandra, Henrik Pedersen, Nick S. Laursen, and Gregers R. Andersen. "Nanobodies Provide Insight into the Molecular Mechanisms of the Complement Cascade and Offer New Therapeutic Strategies." Biomolecules 11, no. 2 (February 17, 2021): 298. http://dx.doi.org/10.3390/biom11020298.

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Abstract:

The complement system is part of the innate immune response, where it provides immediate protection from infectious agents and plays a fundamental role in homeostasis. Complement dysregulation occurs in several diseases, where the tightly regulated proteolytic cascade turns offensive. Prominent examples are atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria and Alzheimer’s disease. Therapeutic intervention targeting complement activation may allow treatment of such debilitating diseases. In this review, we describe a panel of complement targeting nanobodies that allow modulation at different steps of the proteolytic cascade, from the activation of the C1 complex in the classical pathway to formation of the C5 convertase in the terminal pathway. Thorough structural and functional characterization has provided a deep mechanistic understanding of the mode of inhibition for each of the nanobodies. These complement specific nanobodies are novel powerful probes for basic research and offer new opportunities for in vivo complement modulation.

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Journal articles on the topic 'Nanobodies'

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