The targeted delivery of chemotherapeutic drugs is a major challenge in the clinical treatment of cancer. Herein, we constructed a multifunctional DNA nanoplatform as a versatile carrier of the ...highly potent platinum‐based DNA intercalator, 56MESS. In our rational design, 56MESS was efficiently loaded into the double‐bundle DNA tetrahedron through intercalation with the DNA duplex. With the integration of a nanobody that both targets and blocks epidermal growth factor receptor (EGFR), the DNA nanocarriers exhibit excellent selectivity for cells with elevated EGFR expression (a common biomarker related to tumor formation) and combined tumor therapy without obvious systemic toxicity. This DNA‐based platinum‐drug delivery system provides a promising strategy for the treatment of tumors.
A double‐bundle DNA tetrahedron‐based nanoplatform integrating a nanobody, which targets and blocks the epidermal growth factor receptor (EGFR), commonly overexpressed on the surface of tumor cells, and a platinum‐based DNA intercalator (56MESS) was constructed for efficient and targeted tumor therapy.
The activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to ...multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified.
To develop an assay that can differentiate the platelet-binding activity of VWF multimers.
A novel enzyme-linked immunosorbent assay (nanobody-triggered glycoprotein Ib binding assay VWF:GPIbNab) utilizing a VWF-activating nanobody was developed. Recombinant VWF, plasma-derived VWF (pdVWF), and selected gel-filtrated fractions of pdVWF were evaluated for VWF antigen and activity levels. A linear regression model was developed to estimate the specific activity of VWF multimers.
Of the 3 activating nanobodies tested, 6C11 with the lowest activation effect exhibited the highest sensitivity for high-molecular-weight multimers (HMWMs) of VWF. VWF:GPIbNab utilizing 6C11 (VWF:GPIbNab6C11) produced significantly higher activity/antigen ratios for recombinant VWF (>2.0) and HMWM-enriched pdVWF fractions (>2.0) than for pdVWF (∼1.0) or fractions enriched with shorter multimers (<1.0). The differences were much larger than those produced by VWF:GPIbNab utilizing other nanobodies, VWF:GPIbM, VWF:GPIbR, or VWF:CB assays. Linear regression analysis of 5 pdVWF fractions of various multimer sizes produced an estimated specific activity of 2.7 for HMWMs. The analysis attributed >90% of the VWF activity measured by VWF:GPIbNab6C11 to that of HMWMs, which is significantly higher than all other activity assays tested.
The VWF:GPIbNab6C11 assay exhibits higher sensitivity to HMWMs than ristocetin-based and collagen-binding assays. Future studies examining the application of this assay in clinical settings and any associated therapeutic benefit of doing so are warranted.
Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, ...and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.
•GFP nanobody was expressed and purified using prokaryotic and eukaryotic expression systems.•Purified GFP nanobody can be used to identify GFP protein and GFP-tagged protein.•The GFP nanobodies derived from two protein expression systems were found to have different GFP recognition abilities.•MS analysis found that GFP nanobodies from two protein expression systems have different post-translational modifications, which may be a promising development trend for nanobody research.
Single‐domain antibodies (sdAbs), the autonomous variable domains of camelid and shark heavy‐chain antibodies, have many desirable properties as components of biologic drugs. However, their sequences ...may increase the risk of immunogenicity and antidrug antibody (ADA) development in humans, and thus, sdAbs are routinely humanized during development. Here, we review and summarize the available evidence regarding the factors governing immunogenicity of sdAbs and our current state of knowledge of strategies to mitigate immunogenicity risks by humanization. While several sdAb properties, including high homology of camelid VHHs with human IGHV3 gene products, favor low immunogenicity in humans, epitopes absent in the human repertoire including the exposed VH:VL interface may be intrinsically immunogenic. While most clinical trials have demonstrated minimal sdAb immunogenicity, two notable exceptions (the tetrameric DR5‐specific VHH TAS266 and the TNFR1‐specific VH GSK1995057) illustrate that special caution must be taken in identifying preexisting ADAs against highly potent sdAbs. Nonhuman sequence alone does not adequately explain sdAb immunogenicity, as some camelid VHHs are nonimmunogenic while some fully human VHs elicit ADAs. The presence of preexisting ADAs directed against the exposed C‐termini of some sdAbs in a significant proportion of individuals awaits a molecular explanation. Whether sdAb humanization reduces or promotes immunogenicity remains unclear: reduction of nonhuman sequence content at the expense of introducing low‐level aggregation in humanized variants may be counterproductive. Further work will establish thresholds for VHH and VNAR humanization to maximize human sequence content while avoiding loss of binding affinity and/or immunogenicity resulting from aggregation or decreased stability.
Single‐domain antibodies (sdAbs), the autonomous variable domains of camelid and shark heavy‐chain antibodies, are potentially immunogenic in humans. We review factors governing sdAb immunogenicity, the consensus of clinical trials of sdAb drugs, and humanization strategies for reducing immunogenicity risk. While most sdAbs are minimally immunogenic, two notable exceptions illustrate that nonhuman sequence alone does not adequately explain immunogenicity. Other factors, including aggregation and intrinsic sdAb properties, may play important roles.
Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post‐translational modifications in the protein's native ...environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real‐time monitoring of protein expression, and also provides an affinity handle for one‐step protein purification using a suitable affinity reagent. Here, we describe a panel of anti‐GFP and anti‐mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X‐ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody‐sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry‐tag system by using it to purify recombinant human topoisomerase 2α expressed in HEK293F cells. The mCherry‐tag and GFP/YFP‐tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real‐time monitoring of protein expression levels and a high‐efficiency purification step is needed.
PDB Code(s): 7SAH, 7SAI, 7SAJ, 7SAK and 7SAL;
The integrin receptor αMβ2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects ...of αMβ2 signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human αMβ2 headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H αM variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside–inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the αM subunit in an Mg2+-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the αM subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound αMβ2 with iC3b, suggesting that it may represent a novel high-affinity proteinaceous αMβ2-specific agonist. Taken together, our data suggest that the iC3b–αMβ2 complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to αMβ2.
In this study, we demonstrated the first report of a phage-mediated double-nanobody sandwich chemiluminescent enzyme immmunoassay (P-CLISA) for Salmonella Typhimurium (S. Typhimurium) determination. ...Epitope mapping was first conducted for pairing nanobodies, and then soluble and phage-displayed nanobodies were used as detection antibodies in Nb-ELISA and P-ELISA, respectively. Compared with Nb-ELISA, P-ELISA has a 100-fold improvement in sensitivity given the signal amplification mediated by phage. Moreover, a chemiluminescence reaction was applied to replace the traditional chromogenic reaction in the assay, which achieved the detection of S. Typhimurium with an LOD of 3.63 × 103 CFU/mL in a linear range from 5.1 × 103 to 1.2 × 106 CFU/mL. The P-CLISA was successfully applied in actual sample analysis and able to detect fewer than 10 S. Typhimurium cells within 6–8 h of incubation. This study provides guidelines to develop reliable and sensitive double nanobody sandwich immunoassays, which also have great potential for other large molecule monitoring in food samples.
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•This is the first report on anti-S. Typhimurium nanobodies.•A phage-mediated double-nanobody sandwich chemiluminescent enzyme immunoassay was developed for detection of S. Typhimurium.•The P-CLISA was achieved detection of S. Typhimurium with a LOD of 3.63 × 103 CFU/mL.
Antibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their ...limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV.
In the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni-NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests.
For the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed immunoassays eliminate the use of commercial secondary antibodies and shorten detection time. Meanwhile, both assays display great developmental prospect for further commercial production and application.
Nanobodies (Nbs) have arisen as an alternative to conventional antibodies (Abs) and show great potential when used as tools in different biotechnology fields such as diagnostics and therapy. ...Different approaches have been described for the production of Nbs and these methods face new challenges focused on improving yield, affinity, and reducing production costs. This review summarizes these challenges, and also the latest advances in the detection of different kinds of molecules, such as proteins and small molecules, and describes their potential use for noninvasive in vivo imaging and for in vitro assays. Moreover, the unique properties of Nbs are outlined like internalization, size, thermal and chemical stability, affinity, blood clearance, and labeling procedures. Concerning therapeutic applications, we highlight some already reported examples about Nbs being used for the treatment of several diseases such as cancer, neurodegenerative or infectious diseases among others. Finally, future trends, opportunities, and disadvantages are also discussed.
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