Research

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16 pages, 2261 KiB

Open AccessCommunication

Single Domain Antibodies as Carriers for Intracellular Drug Delivery: A Proof of Principle Study

by Sebas D. Pronk, Erik Schooten, Jurgen Heinen, Esra Helfrich, Sabrina Oliveira and Paul M. P. van Bergen en Henegouwen

Biomolecules 2021, 11(7), 927; https://doi.org/10.3390/biom11070927 - 22 Jun 2021

Cited by 2 | Viewed by 3373

Abstract

Antibody-drug conjugates (ADCs) are currently used for the targeted delivery of drugs to diseased cells, but intracellular drug delivery and therefore efficacy may be suboptimal because of the large size, slow internalization and ineffective intracellular trafficking of the antibody. Using a phage display [...] Read more.

Antibody-drug conjugates (ADCs) are currently used for the targeted delivery of drugs to diseased cells, but intracellular drug delivery and therefore efficacy may be suboptimal because of the large size, slow internalization and ineffective intracellular trafficking of the antibody. Using a phage display method selecting internalizing phages only, we developed internalizing single domain antibodies (sdAbs) with high binding affinity to rat PDGFRβ, a receptor involved in different types of diseases. We demonstrate that these constructs have different characteristics with respect to internalization rates but all traffic to lysosomes. To compare their efficacy in targeted drug delivery, we conjugated the sdAbs to a cytotoxic drug. The conjugates showed improved cytotoxicity correlating to their internalization speed. The efficacy of the conjugates was inhibited in the presence of vacuolin-1, an inhibitor of lysosomal maturation, suggesting lysosomal trafficking is needed for efficient drug release. In conclusion, sdAb constructs with different internalization rates can be designed against the same target, and sdAbs with a high internalization rate induce more cell killing than sdAbs with a lower internalization rate in vitro. Even though the overall efficacy should also be tested in vivo, sdAbs are particularly interesting formats to be explored to obtain different internalization rates. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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14 pages, 2667 KiB

Open AccessArticle

The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

by Pieterjan Debie, Noemi B. Declerck, Danny van Willigen, Celine M. Huygen, Bieke De Sloovere, Lukasz Mateusiak, Jessica Bridoux, Janik Puttemans, Nick Devoogdt, Fijs W. B. van Leeuwen and Sophie Hernot

Biomolecules 2021, 11(3), 360; https://doi.org/10.3390/biom11030360 - 26 Feb 2021

Cited by 8 | Viewed by 2486

Abstract

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, [...] Read more.

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and ¹¹¹In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [¹¹¹In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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15 pages, 2423 KiB

Open AccessArticle

Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer

by Markus Kilisch, Hansjörg Götzke, Maja Gere-Becker, Alexander Crauel, Felipe Opazo and Steffen Frey

Biomolecules 2021, 11(2), 269; https://doi.org/10.3390/biom11020269 - 12 Feb 2021

Cited by 2 | Viewed by 4775

Abstract

Epitope tags are widely employed as tools to detect, purify and manipulate proteins in various experimental systems. We recently introduced the ALFA-tag together with two ALFA-specific single-domain antibodies (sdAbs), NbALFA and NbALFA^PE, featuring high or intermediate affinity, respectively. Together, the ALFA [...] Read more.

Epitope tags are widely employed as tools to detect, purify and manipulate proteins in various experimental systems. We recently introduced the ALFA-tag together with two ALFA-specific single-domain antibodies (sdAbs), NbALFA and NbALFA^PE, featuring high or intermediate affinity, respectively. Together, the ALFA system can be employed for a broad range of applications in microscopy, cell biology and biochemistry requiring either extraordinarily stable binding or mild competitive elution at room temperature. In order to further enhance the versatility of the ALFA system, we, here, aimed at developing an sdAb optimized for efficient elution at low temperatures. To achieve this, we followed a stringent selection scheme tailored to the specific application. We found candidates combining a fast capture of ALFA-tagged proteins with an efficient competitive elution at 4 °C in physiological buffer. Importantly, by employing a structure-guided semisynthetic library based on well-characterized NbALFA variants, the high specificity and consistent binding of proteins harboring ALFA-tags at either terminus could be maintained. ALFA Selector^CE, a resin presenting the cold-elutable NbALFA^CE, is an ideal tool for the one-step purification of sensitive protein complexes or temperature-labile enzymes. We believe that the general approach followed during the selection and screening can be transferred to other challenging sdAb discovery projects. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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13 pages, 6967 KiB

Open AccessArticle

Nanobody-Based Indirect Competitive ELISA for Sensitive Detection of 19-Nortestosterone in Animal Urine

by Yuan-yuan Yang, Yu Wang, Yi-feng Zhang, Feng Wang, Yi-fan Liang, Jin-yi Yang, Zhen-lin Xu, Yu-dong Shen and Hong Wang

Biomolecules 2021, 11(2), 167; https://doi.org/10.3390/biom11020167 - 27 Jan 2021

Cited by 6 | Viewed by 2763

Abstract

Nanobody (Nb), a new type of biorecognition element generally from Camelidae, has the characteristics of small molecular weight, high stability, great solubility and high expression level in E. coli. In this study, with 19-nortestosterone (19-NT), an anabolic androgenic steroid as target [...] Read more.

Nanobody (Nb), a new type of biorecognition element generally from Camelidae, has the characteristics of small molecular weight, high stability, great solubility and high expression level in E. coli. In this study, with 19-nortestosterone (19-NT), an anabolic androgenic steroid as target drug, three specific Nbs against 19-NT were selected from camel immune library by phage display technology. The obtained Nbs showed excellent thermostability and organic solvent tolerance. The nanobody Nb2F7 with the best performance was used to develop a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for 19-NT detection. Under optimized conditions, the standard curve of ic-ELISA was fitted with a half-maximal inhibitory concentration (IC₅₀) of 1.03 ng/mL and a detection limit (LOD) of 0.10 ng/mL for 19-NT. Meanwhile, the developed assay had low cross- reactivity with analogs and the recoveries of 19-NT ranged from 82.61% to 99.24% in spiked samples. The correlation coefficient between ic-ELISA and the ultra-performance liquid chromatography/mass spectrometry (UPLC-MS/MS) method was 0.9975, which indicated that the nanobody-based ic-ELISA could be a useful tool for a rapid analysis of 19-NT in animal urine samples. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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13 pages, 2048 KiB

Open AccessArticle

Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region

by Miguel A. Soler, Barbara Medagli, Jiewen Wang, Sandra Oloketuyi, Gregor Bajc, He Huang, Sara Fortuna and Ario de Marco

Biomolecules 2021, 11(2), 163; https://doi.org/10.3390/biom11020163 - 26 Jan 2021

Cited by 13 | Viewed by 4510

Abstract

In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized [...] Read more.

In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the Camelidae hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability. We described at molecular level how the interaction among residues belonging to different structural elements enabled a model llama nanobody (C8WT, isolated from a naïve library) to be functional and maintain its stability, despite the analysis of its primary sequence would classify it as aggregation-prone. Five chimeras formed by grafting CDRs isolated from different nanobodies into C8WT scaffold were successfully expressed as soluble proteins and both tested clones preserved their antigen binding specificity. We identified a nanobody with human hallmarks that seems suitable for humanizing selected camelid VHHs by grafting heterologous CDRs in its scaffold and could serve for the preparation of a synthetic library of human-like single domains. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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11 pages, 1478 KiB

Open AccessArticle

Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs

by Ainhoa Moliner-Morro, Daniel J. Sheward, Vivien Karl, Laura Perez Vidakovics, Ben Murrell, Gerald M. McInerney and Leo Hanke

Biomolecules 2020, 10(12), 1661; https://doi.org/10.3390/biom10121661 - 11 Dec 2020

Cited by 24 | Viewed by 4881

Abstract

Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of [...] Read more.

Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC₅₀ in the low picomolar range. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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14 pages, 1753 KiB

Open AccessArticle

Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA

by Triana Delfin-Riela, Martín Rossotti, Romina Alvez-Rosado, Carmen Leizagoyen and Gualberto González-Sapienza

Biomolecules 2020, 10(12), 1652; https://doi.org/10.3390/biom10121652 - 09 Dec 2020

Cited by 17 | Viewed by 2366

Abstract

The Zika virus was introduced in Brazil in 2015 and, shortly after, spread all over the Americas. Nowadays, it remains present in more than 80 countries and represents a major threat due to some singularities among other flaviviruses. Due to its easy transmission, [...] Read more.

The Zika virus was introduced in Brazil in 2015 and, shortly after, spread all over the Americas. Nowadays, it remains present in more than 80 countries and represents a major threat due to some singularities among other flaviviruses. Due to its easy transmission, high percentage of silent cases, the severity of its associated complications, and the lack of prophylactic methods and effective treatments, it is essential to develop reliable and rapid diagnostic tests for early containment of the infection. Nonstructural protein 1 (NS1), a glycoprotein involved in all flavivirus infections, is secreted since the beginning of the infection into the blood stream and has proven to be a valuable biomarker for the early diagnosis of other flaviviral infections. Here, we describe the development of a highly sensitive nanobody ELISA for the detection of the NS1 protein in serum samples. Nanobodies were selected from a library generated from a llama immunized with Zika NS1 (ZVNS1) by a two-step high-throughput screening geared to identify the most sensitive and specific nanobody pairs. The assay was performed with a sub-ng/mL detection limit in the sera and showed excellent reproducibility and accuracy when validated with serum samples spiked with 0.80, 1.60, or 3.10 ng/mL of ZVNS1. Furthermore, the specificity of the developed ELISA was demonstrated using a panel of flavivirus’ NS1 proteins; this is of extreme relevance in countries endemic for more than one flavivirus. Considering that the nanobody sequences are provided, the assay can be reproduced in any laboratory at low cost, which may help to strengthen the diagnostic capacity of the disease even in low-resource countries. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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12 pages, 1440 KiB

Open AccessArticle

Peptide Linker Affecting the Activity Retention Rate of VHH in Immunosorbents

by Da Li, Jun Ren, Fangling Ji, Qiang Peng, Hu Teng and Lingyun Jia

Biomolecules 2020, 10(12), 1610; https://doi.org/10.3390/biom10121610 - 27 Nov 2020

Cited by 4 | Viewed by 2881

Abstract

VHH-based immunosorbents are an emerging and promising tool for the removal of toxic substances from plasma. However, the small size of VHHs is a double-edged sword, bringing both benefits and drawbacks to the immunosorbent. The small size of the VHH allows a higher [...] Read more.

VHH-based immunosorbents are an emerging and promising tool for the removal of toxic substances from plasma. However, the small size of VHHs is a double-edged sword, bringing both benefits and drawbacks to the immunosorbent. The small size of the VHH allows a higher coupling density, while the closer distance to the resin might create steric hindrance for paratope access. The latter could be avoided by inserting a linker between the VHH and the gel attachment site. Here, we report an approach to improve the activity retention of the immobilized VHH by selecting suitable linkers between the VHH and the site-specific immobilization site on the resin. Seven peptide linkers differing in length and flexibility were fused to the VHH and contained the formylglycine generating enzyme (FGE) recognition sequence. These constructs were expressed in the cytoplasm of bacteria and purified, the VHH production yield and affinity for its cognate antigen was measured. Furthermore, the fGly conversion, the immobilization of the aldehyde-containing nanobodies, the immobilization on resin and the antigen binding activity of the VHH-based immunoadsorbents was monitored. The VHH with longer and rigid, proline-rich linkers exhibited good expression yield of approximately 160 mg/L of culture, a fGly conversion of up to 100%, and the highest activity retention rate of more than 68%. This study unveiled two suitable linkers for the preparation of VHH-based immunosorbents that will assist the development of their clinical application. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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15 pages, 1290 KiB

Open AccessFeature PaperArticle

Anti-Human PD-L1 Nanobody for Immuno-PET Imaging: Validation of a Conjugation Strategy for Clinical Translation

by Jessica Bridoux, Katrijn Broos, Quentin Lecocq, Pieterjan Debie, Charlotte Martin, Steven Ballet, Geert Raes, Sara Neyt, Christian Vanhove, Karine Breckpot, Nick Devoogdt, Vicky Caveliers, Marleen Keyaerts and Catarina Xavier

Biomolecules 2020, 10(10), 1388; https://doi.org/10.3390/biom10101388 - 29 Sep 2020

Cited by 42 | Viewed by 4508

Abstract

Immune checkpoints, such as programmed death-ligand 1 (PD-L1), limit T-cell function and tumor cells use this ligand to escape the anti-tumor immune response. Treatments with monoclonal antibodies blocking these checkpoints have shown long-lasting responses, but only in a subset of patients. This study [...] Read more.

Immune checkpoints, such as programmed death-ligand 1 (PD-L1), limit T-cell function and tumor cells use this ligand to escape the anti-tumor immune response. Treatments with monoclonal antibodies blocking these checkpoints have shown long-lasting responses, but only in a subset of patients. This study aims to develop a Nanobody (Nb)-based probe in order to assess human PD-L1 (hPD-L1) expression using positron emission tomography imaging, and to compare the influence of two different radiolabeling strategies, since the Nb has a lysine in its complementarity determining region (CDR), which may impact its affinity upon functionalization. The Nb has been conjugated with the NOTA chelator site-specifically via the Sortase-A enzyme or randomly on its lysines. [⁶⁸Ga]Ga-NOTA-(hPD-L1) Nbs were obtained in >95% radiochemical purity. In vivo tumor targeting studies at 1 h 20 post-injection revealed specific tumor uptake of 1.89 ± 0.40%IA/g for the site-specific conjugate, 1.77 ± 0.29%IA/g for the random conjugate, no nonspecific organ targeting, and excretion via the kidneys and bladder. Both strategies allowed for easily obtaining ⁶⁸Ga-labeled hPD-L1 Nbs in high yields. The two conjugates were stable and showed excellent in vivo targeting. Moreover, we proved that the random lysine-conjugation is a valid strategy for clinical translation of the hPD-L1 Nb, despite the lysine present in the CDR. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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Review

Jump to: Research

32 pages, 6622 KiB

Open AccessReview

Nanobodies for Medical Imaging: About Ready for Prime Time?

by Léa Berland, Lauren Kim, Omar Abousaway, Andrea Mines, Shruti Mishra, Louise Clark, Paul Hofman and Mohammad Rashidian

Biomolecules 2021, 11(5), 637; https://doi.org/10.3390/biom11050637 - 26 Apr 2021

Cited by 21 | Viewed by 10241

Abstract

Recent advances in medical treatments have been revolutionary in shaping the management and treatment landscape of patients, notably cancer patients. Over the last decade, patients with diverse forms of locally advanced or metastatic cancer, such as melanoma, lung cancers, and many blood-borne malignancies, [...] Read more.

Recent advances in medical treatments have been revolutionary in shaping the management and treatment landscape of patients, notably cancer patients. Over the last decade, patients with diverse forms of locally advanced or metastatic cancer, such as melanoma, lung cancers, and many blood-borne malignancies, have seen their life expectancies increasing significantly. Notwithstanding these encouraging results, the present-day struggle with these treatments concerns patients who remain largely unresponsive, as well as those who experience severely toxic side effects. Gaining deeper insight into the cellular and molecular mechanisms underlying these variable responses will bring us closer to developing more effective therapeutics. To assess these mechanisms, non-invasive imaging techniques provide valuable whole-body information with precise targeting. An example of such is immuno-PET (Positron Emission Tomography), which employs radiolabeled antibodies to detect specific molecules of interest. Nanobodies, as the smallest derived antibody fragments, boast ideal characteristics for this purpose and have thus been used extensively in preclinical models and, more recently, in clinical early-stage studies as well. Their merit stems from their high affinity and specificity towards a target, among other factors. Furthermore, their small size (~14 kDa) allows them to easily disperse through the bloodstream and reach tissues in a reliable and uniform manner. In this review, we will discuss the powerful imaging potential of nanobodies, primarily through the lens of imaging malignant tumors but also touching upon their capability to image a broader variety of nonmalignant diseases. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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22 pages, 5263 KiB

Open AccessReview

Broad Reactivity Single Domain Antibodies against Influenza Virus and Their Applications to Vaccine Potency Testing and Immunotherapy

by Andrew Tung Yep, Yasu Takeuchi, Othmar G. Engelhardt and Simon E. Hufton

Biomolecules 2021, 11(3), 407; https://doi.org/10.3390/biom11030407 - 10 Mar 2021

Cited by 6 | Viewed by 4134

Abstract

The antigenic variability of influenza presents many challenges to the development of vaccines and immunotherapeutics. However, it is apparent that there are epitopes on the virus that have evolved to remain largely constant due to their functional importance. These more conserved regions are [...] Read more.

The antigenic variability of influenza presents many challenges to the development of vaccines and immunotherapeutics. However, it is apparent that there are epitopes on the virus that have evolved to remain largely constant due to their functional importance. These more conserved regions are often hidden and difficult to access by the human immune system but recent efforts have shown that these may be the Achilles heel of the virus through development and delivery of appropriate biological drugs. Amongst these, single domain antibodies (sdAbs) are equipped to target these vulnerabilities of the influenza virus due to their preference for concave epitopes on protein surfaces, their small size, flexible reformatting and high stability. Single domain antibodies are well placed to provide a new generation of robust analytical reagents and therapeutics to support the constant efforts to keep influenza in check. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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15 pages, 740 KiB

Open AccessReview

Unique Benefits of Tumor-Specific Nanobodies for Fluorescence Guided Surgery

by Thinzar M. Lwin, Robert M. Hoffman and Michael Bouvet

Biomolecules 2021, 11(2), 311; https://doi.org/10.3390/biom11020311 - 18 Feb 2021

Cited by 6 | Viewed by 3088

Abstract

Tumor-specific fluorescence labeling is promising for real-time visualization of solid malignancies during surgery. There are a number of technologies to confer tumor-specific fluorescence. Antibodies have traditionally been used due to their versatility in modifications; however, their large size hampers efficient fluorophore delivery. Nanobodies [...] Read more.

Tumor-specific fluorescence labeling is promising for real-time visualization of solid malignancies during surgery. There are a number of technologies to confer tumor-specific fluorescence. Antibodies have traditionally been used due to their versatility in modifications; however, their large size hampers efficient fluorophore delivery. Nanobodies are a novel class of molecules, derived from camelid heavy-chain only antibodies, that have shown promise for tumor-specific fluorescence labeling. Nanobodies are ten times smaller than standard antibodies, while maintaining antigen-binding capacity and have advantageous features, including rapidity of tumor labeling, that are reviewed in the present report. The present report reviews special considerations needed in developing nanobody probes, the status of current literature on the use of nanobody probes in fluorescence guided surgery, and potential challenges to be addressed for clinical translation. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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23 pages, 4709 KiB

Open AccessReview

Nanobodies Provide Insight into the Molecular Mechanisms of the Complement Cascade and Offer New Therapeutic Strategies

by Alessandra Zarantonello, Henrik Pedersen, Nick S. Laursen and Gregers R. Andersen

Biomolecules 2021, 11(2), 298; https://doi.org/10.3390/biom11020298 - 17 Feb 2021

Cited by 5 | Viewed by 4134

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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18 pages, 1427 KiB

Open AccessReview

The Application of Nanobody in CAR-T Therapy

by Chaolemeng Bao, Quanli Gao, Lin-Lin Li, Lu Han, Bingxiang Zhang, Yijin Ding, Zongpei Song, Ruining Zhang, Jishuai Zhang and Xian-Hui Wu

Biomolecules 2021, 11(2), 238; https://doi.org/10.3390/biom11020238 - 08 Feb 2021

Cited by 45 | Viewed by 8201

Abstract

Chimeric antigen receptor (CAR) T therapy represents a form of immune cellular therapy with clinical efficacy and a specific target. A typical chimeric antigen receptor (CAR) construct consists of an antigen binding domain, a transmembrane domain, and a cytoplasmic domain. Nanobodies have been [...] Read more.

Chimeric antigen receptor (CAR) T therapy represents a form of immune cellular therapy with clinical efficacy and a specific target. A typical chimeric antigen receptor (CAR) construct consists of an antigen binding domain, a transmembrane domain, and a cytoplasmic domain. Nanobodies have been widely applied as the antigen binding domain of CAR-T due to their small size, optimal stability, high affinity, and manufacturing feasibility. The nanobody-based CAR structure has shown a proven function in more than ten different tumor-specific targets. After being transduced in Jurkat cells, natural killer cells, or primary T cells, the resulting nanobody-based CAR-T or CAR-NK cells demonstrate anti-tumor effects both in vitro and in vivo. Interestingly, anti-BCMA CAR-T modulated by a single nanobody or bi-valent nanobody displays comparable clinical effects with that of single-chain variable fragment (scFv)-modulated CAR-T. The application of nanobodies in CAR-T therapy has been well demonstrated from bench to bedside and displays great potential in forming advanced CAR-T for more challenging tasks. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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19 pages, 1717 KiB

Open AccessReview

Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor

by Raf Van Campenhout, Serge Muyldermans, Mathieu Vinken, Nick Devoogdt and Timo W.M. De Groof

Biomolecules 2021, 11(1), 63; https://doi.org/10.3390/biom11010063 - 06 Jan 2021

Cited by 14 | Viewed by 4554

Abstract

Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the [...] Read more.

Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the cytosol. A plethora of human pathologies are associated with the altered expression or dysfunction of cell plasma membrane transport proteins, making them interesting therapeutic drug targets. However, the search for therapeutics is challenging, since many drug candidates targeting cell plasma membrane proteins fail in (pre)clinical testing due to inadequate selectivity, specificity, potency or stability. These latter characteristics are met by nanobodies, which potentially renders them eligible therapeutics targeting cell plasma membrane proteins. Therefore, a therapeutic nanobody-based strategy seems a valid approach to target and modulate the activity of cell plasma membrane transport proteins. This review paper focuses on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discusses several therapeutic nanobodies directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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18 pages, 1208 KiB

Open AccessEditor’s ChoiceReview

Nanobodies Right in the Middle: Intrabodies as Toolbox to Visualize and Modulate Antigens in the Living Cell

by Teresa R. Wagner and Ulrich Rothbauer

Biomolecules 2020, 10(12), 1701; https://doi.org/10.3390/biom10121701 - 21 Dec 2020

Cited by 21 | Viewed by 4989

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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24 pages, 1931 KiB

Open AccessReview

Nanobodies as Versatile Tool for Multiscale Imaging Modalities

by Marco Erreni, Tilo Schorn, Francesca D’Autilia and Andrea Doni

Biomolecules 2020, 10(12), 1695; https://doi.org/10.3390/biom10121695 - 18 Dec 2020

Cited by 17 | Viewed by 3309

Abstract

Molecular imaging is constantly growing in different areas of preclinical biomedical research. Several imaging methods have been developed and are continuously updated for both in vivo and in vitro applications, in order to increase the information about the structure, localization and function of [...] Read more.

Molecular imaging is constantly growing in different areas of preclinical biomedical research. Several imaging methods have been developed and are continuously updated for both in vivo and in vitro applications, in order to increase the information about the structure, localization and function of molecules involved in physiology and disease. Along with these progresses, there is a continuous need for improving labeling strategies. In the last decades, the single domain antigen-binding fragments nanobodies (Nbs) emerged as important molecular imaging probes. Indeed, their small size (~15 kDa), high stability, affinity and modularity represent desirable features for imaging applications, providing higher tissue penetration, rapid targeting, increased spatial resolution and fast clearance. Accordingly, several Nb-based probes have been generated and applied to a variety of imaging modalities, ranging from in vivo and in vitro preclinical imaging to super-resolution microscopy. In this review, we will provide an overview of the state-of-the-art regarding the use of Nbs in several imaging modalities, underlining their extreme versatility and their enormous potential in targeting molecules and cells of interest in both preclinical and clinical studies. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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An Inside Job: Applications of Intracellular Single Domain Antibodies

by Eline Soetens, Marlies Ballegeer and Xavier Saelens

Biomolecules 2020, 10(12), 1663; https://doi.org/10.3390/biom10121663 - 12 Dec 2020

Cited by 16 | Viewed by 4002

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue The Therapeutic and Diagnostic Potential of Nanobodies)

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