Acute renal failure (ARF) represents a very important and potentially devastating disorder in clinical nephrology. Neutrophil gelatinase-associated lipocalin (NGAL) is an early biomarker for ARF in a ...wide range of different disease processes, which is frequently detected in clinical diagnosis. Herein, we present a label-free and sensitive photoelectrochemical (PEC) immunosensor for NGAL by utilizing a biotinylated anti-NGAL Nanobody (Nb) orientedly immobilized to streptavidin-coated cobalt 2,9,16,23-tetraaminophthalocyanine (CoPc)-sensitized TiO2 electrode. The Nb was biotinylated at the C-terminus, which is situated at the opposite site of the antigen binding region. Using highly oriented Nb as receptor molecules, a label-free PEC immunosensor for NGAL was developed by monitoring the changes in the photocurrent signals of the electrode resulting from immunoreaction. Immobilization of Nb to streptavidin-coated CoPc-sensitized TiO2 electrode surface provides high binding capacity to NGAL; thus, it can lead to a high sensitivity. The limit of detection (LOD) of the proposed immunosensor has been significantly lowered to 0.6 pg mL(-1). This proposed immunosensor reveals high specificity to detect NGAL, with acceptable intra-assay precision and excellent stability. In addition, the present work provides a new approach to design Nb-based PEC immunosensor and increases versatility of Nbs.
Purpose
Overexpression of epithelial cell adhesion molecule (EpCAM) plays essential roles in tumorigenesis and tumor progression in almost all epithelium-derived cancer. Monitoring EpCAM expression ...in tumors can be used for the diagnosis, staging, and prognosis of cancer patients, as well as guiding the individualized treatment of EpCAM-targeted drugs. In this study, we described the synthesis and evaluation of a site-specifically
99m
TcTc-labeled EpCAM-targeted nanobody for the SPECT/CT imaging of EpCAM expression.
Methods
We first prepared the
99m
TcTc-HYNIC-G
4
K; then, it was site-specifically connected to EpCAM-targeted nanobody NB4. The in vitro characteristics of
99m
TcTc-NB4 were investigated in HT-29 (EpCAM positive) and HL-60 (EpCAM negative) cells, while the in vivo studies were performed using small-animal SPECT/CT in the subcutaneous tumor models and the lymph node metastasis model to verify the specific targeting capacity as well as the potential applications of
99m
TcTc-NB4.
Results
99m
TcTc-NB4 displayed a high EpCAM specificity both in vitro and in vivo. SPECT/CT imaging revealed that
99m
TcTc-NB4 was cleared rapidly from the blood and normal organs except for the kidneys, and HT-29 tumors were clearly visualized in contrast with HL-60 tumors. The uptake value of
99m
TcTc-NB4 in HT-29 tumors was increased continuously from 3.77 ± 0.39%ID/g at 0.5 h to 5.53 ± 0.82%ID/g at 12 h after injection. Moreover, the
99m
TcTc-NB4 SPECT/CT could clearly image tumor-draining lymph nodes.
Conclusion
99m
TcTc-NB4 is a broad-spectrum, specific, and sensitive SPECT radiotracer for the noninvasive imaging of EpCAM expression in the epithelium-derived cancer and revealed a great potential for the clinical translation.
CD47, the integrin-related protein, plays an important role in immune resistance and escape of tumor cells. Antibodies blocking the CD47/SIRPα signal pathway can effectively stimulate ...macrophage-mediated phagocytosis of tumor cells, which becomes a promising approach for tumor immunotherapy. Nanobodies (Nbs) derived from camelid animals are emerging as a new force in antibody therapy.
HuNb1-IgG4, an innovative anti-CD47 nanobody, was developed with high affinity and specificity. It effectively enhanced macrophage-mediated phagocytosis of tumor cells in vitro and showed potent anti-ovarian and anti-lymphoma activity in vivo. Importantly, HuNb1-IgG4 did not induce the agglutination of human red blood cells (RBCs) in vitro and exhibited high safety for hematopoietic system in cynomolgus monkey. In addition, HuNb1-IgG4 could be produced on a large scale in CHO-S cells with high activity and good stability. Also, we established anti-CD47/CD20 bispecific antibody (BsAb) consisted of HuNb1 and Rituximab, showing more preference binding to tumor cells and more potent anti-lymphoma activity compared to HuNb1-IgG4.
Both of HuNb1-IgG4 and anti-CD47/CD20 BsAb are potent antagonists of CD47/SIRPα pathway and promising candidates for clinical trials.
Influenza H5N1 is one subtype of the influenza A virus which can infect human bodies and lead to death. Timely diagnosis before its breakout is vital to the human health. The current clinical ...biochemical diagnosis for influenza virus are still flawed, and the diagnostic kits of H5N1 are mainly based on traditional monoclonal antibodies that hardly meet the requirements for clinical applications. Nanobody is a promising tool for diagnostics and treatment due to its smallest size, high specificity and stability. In this study, a novel Nanobody-based bioassay was developed for rapid, low-cost and sensitive detection of the influenza H5N1 virus.
Nanobodies specific to H5N1 virus were selected from a VHH library by phage display technology. In this system, the biotinylated Nanobody was directionally captured by streptavidin coated on ELISA plate, which can specifically capture the H5N1 virus. Another Nanobody conjugated with HRP was used as a detector. A novel directional enzyme-linked immunosorbent assay for H5N1 using specific Nanobodies was established and compared to the conventional undirected ELISA assay.
We have successfully constructed a high quality phage display Nanobody library and isolated two Nanobodies against H5N1 with high affinity and specificity. These two Nanobodies were further used to prepare the biosensor detection system. This streptavidin-biotin-based directional double Nanobodies sandwich ELISA for H5N1 detection showed superiority over the commonly undirectional ELISA protocol. The linear range of detection for standards in this immunoassay was approximately 50-1000 ng/mL and the detection limit was 14.1 ng/mL. The average recoveries of H5N1 virus from human serum samples were in the range from 94.58% to 114.51%, with a coefficient of variation less than 6.5%.
Collectively, these results demonstrated that the proposed detection system is an alternative diagnostic tool that enables a rapid, inexpensive, sensitive and specific detection of the influenza virus.
Nanobodies, derived from camelid heavy-chain antibodies, have novel and impactful applications in clinical diagnostics. Our objective is to develop a nanobody-based chemiluminescence immunoassay for ...sensitive detection of human prealbumin (PA). In this context, a phage display nanobody library is constructed via immunizing dromedary camel with human prealbumin. Three nanobodies have been identified by five successive bio-panning steps. Based on their high expression level and good affinity, two out of three are chosen for further study. Magnetic beads (MBs) were functionalized with PEI by acylamide bond formed between the carboxyl group on the surface of the MB. Then, an anti-PA nanobody (Nb1) can be effectively immobilized onto the surface of the functionalized MB using glutaradehyde as the link. The modified MBs with Nb1 can specifically capture the target PA and reacted with silica nanoparticles with co-immobilized HRP and anti-PA nanobody (Nb2). The concentration of PA was detected by flow injection chemiluminescence. When using MB/PEI as the carrier of anti-PA Nb1, the CL signal significantly increased to 4-fold compared with the signal using MB without PEI modification. The CL signal was further amplified to 5-fold when Si/Nb2 was used as the signal probe. Under optimized conditions, the present immunoassay exhibited a wide quantitative range from 0.05 to 1000μgL−1 with a detection limit of 0.01μgL−1. The sensitivity of the proposed immunoassay offers great promises in providing a sensitive, specific, time saving, and potential method for detecting PA in clinical settings.
•Nanobodies against human prealbumin have been first isolated.•MB/PEI/Nb1 was used for capturing prealbumin.•HRP-coated Si/Nb2 was used for chemiluminescence detection.•The reported limit of detection for PA is LOD=0.01ugL−1.•Immunoassay exhibited a wide quantitative range from 0.05 to 1000μgL−1.
PD-1/PD-L1 pathway blocking with antibodies offers a vital and efficient therapeutic strategy to restore T cell-associated antitumor immunity and treats a variety of cancers in clinic. Nanobodies ...(Nbs) give several advantages over conventional monoclonal antibodies such as size, solubility, stability and costs. Additionally, P. pastoris is a suitable host for Nb production. Herein, we aim to produce and evaluate anti-PD-1 Nb derived from the P. pastoris. Our findings indicated that we successfully established the Nbs phage-displayed library against PD-1 with qualified library capacity and insert ratio. Anti-PD-1 Nb Nb97 was screened through PE-ELISA and flow cytometry. To extend half-life of Nb97, we contracted pPICZɑA-Nb97-Nb97-HSA recombination vector, which was then transformed into the system of P. pastoris X-33. The yield of purified Nb97–Nb97-Human serum albumin (HSA) fused protein (MY2935) reached to 2.3 g/L after 147 h of fermentation. Meanwhile, the blocking effect of MY2935 is similar to that of MY2626 (humanized Nb97-Fc), and MY2935 showed better performance on stimulating the immune function through PD-1 reporter assay. Hence, P. pastoris X-33 expressing and secreting functional anti-PD-1 Nb-HSA fusion protein might be a system of high yield and low cost.
•A novel nanobody (Nb) targeting PD-1 was obtained based on the camel VHH framework.•Anti-PD-1 Nb-HSA fusion protein was designed and produced to improve the druggability of anti-PD-1 Nb.•Anti-PD-1 Nb-HSA showed potent blocking activity and could be produced on a large scale with low cost in P. pastoris X-33.
The development of a nanobody-based electrochemiluminescent immunosensor for procalcitonin quantification is described. A highly specific and enhanced sensitivity of target detection was achieved by ...CdTe quantum dot encapsulated silica nanoparticle-assisted signal amplification.
Embedded in the nuclear envelope, nuclear pore complexes (NPCs) not only regulate nuclear transport but also interface with transcriptionally active euchromatin, largely silenced heterochromatin, as ...well as the boundaries between these regions. It is unclear what functional role NPCs play in establishing or maintaining these distinct chromatin domains. We report that the yeast NPC protein Nup170p interacts with regions of the genome that contain ribosomal protein and subtelomeric genes, where it functions in nucleosome positioning and as a repressor of transcription. We show that the role of Nup170p in subtelomeric gene silencing is linked to its association with the RSC chromatin-remodeling complex and the silencing factor Sir4p, and that the binding of Nup170p and Sir4p to subtelomeric chromatin is cooperative and necessary for the association of telomeres with the nuclear envelope. Our results establish the NPC as an active participant in silencing and the formation of peripheral heterochromatin.
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► Nup170p physically interacts with Sir4p, Rap1p, and the chromatin remodeler RSC ► Nup170p binds and represses expression of subtelomeric and ribosomal protein genes ► Nup170p and Sir4p bind cooperatively to subtelomeric DNA ► Tethering of subtelomeric chromatin to the NE during G1 phase requires Nup170p
The nuclear pore complex protein Nup170p associates with specific regions of the yeast genome via interactions with the silencing factor Sir4p. Nup170p represses gene transcription, regulates nucleosome positioning, and promotes telomere tethering to the nuclear envelope, suggesting a role in gene silencing and chromatin structure.
The coronavirus disease 2019 (COVID‐19) pandemic has become a serious burden on global public health. Although therapeutic drugs against COVID‐19 have been used in many countries, their efficacy is ...still limited. We here reported nanobody (Nb) phage display libraries derived from four camels immunized with the SARS‐CoV‐2 spike receptor‐binding domain (RBD), from which 381 Nbs were identified to recognize SARS‐CoV‐2‐RBD. Furthermore, seven Nbs were shown to block interaction of human angiotensin‐converting enzyme 2 (ACE2) with SARS‐CoV‐2‐RBD variants and two Nbs blocked the interaction of human ACE2 with bat‐SL‐CoV‐WIV1‐RBD and SARS‐CoV‐1‐RBD. Among these candidates, Nb11‐59 exhibited the highest activity against authentic SARS‐CoV‐2 with 50% neutralizing dose (ND50) of 0.55 μg/ml. Nb11‐59 can be produced on large scale in Pichia pastoris, with 20 g/L titer and 99.36% purity. It also showed good stability profile, and nebulization did not impact its stability. Overall, Nb11‐59 might be a promising prophylactic and therapeutic molecule against COVID‐19, especially through inhalation delivery.
We reported nanobody (Nb) phage display libraries derived from four camels immunized with the SARS‐CoV‐2 spike receptor‐binding domain (RBD), from which 381 Nbs were identified to recognize SARS‐CoV‐2‐RBD including several mutants. Nb11‐59 exhibited potent antiviral activity against authentic SARS‐CoV‐2 with 50% neutralizing dose (ND50) of 0.55 μg/ml, and it can be produced on large scale in Pichia pastoris with titers reached 20 g/L. Importantly, Nb11‐59 showed a good stability and could be developed as an inhaled drug to treat COVID‐19.
Gastric cancer represents a highly lethal malignancy with an elevated mortality rate among cancer patients, coupled with a suboptimal postoperative survival prognosis. Nectin-4, an overexpressed ...oncological target for various cancers, has been exploited to create antibody-drug conjugates (ADCs) to treat solid tumors. However, there is limited research on Nectin-4 ADCs specifically for gastric cancer, and conventional immunoglobulin G (IgG)-based ADCs frequently encounter binding site barriers. Based on the excellent tumor penetration capabilities inherent in nanobodies (Nbs), we developed Nectin-4-targeting Nb drug conjugates (NDCs) for the treatment of gastric cancer.
An immunized phage display library was established and employed for the selection of Nectin-4-specific Nbs using phage display technology. Subsequently, these Nbs were engineered into homodimers to enhance Nb affinity. To prolong in vivo half-life and reduce immunogenicity, we fused an Nb targeting human serum albumin (HSA), resulting in the development of trivalent humanized Nbs. Further, we site-specifically conjugated a monomethyl auristatin E (MMAE) at the C-terminus of the trivalent Nbs, creating Nectin-4 NDC (huNb26/Nb26-Nbh-MMAE) with a drug-to-antibody ratio (DAR) of 1. Nectin-4 NDC demonstrated excellent in vitro cell-binding activities and cytotoxic efficacy against cells with high Nectin-4 expression. Subsequent administration of Nectin-4 NDC to mice bearing NCI-N87 human gastric cancer xenografts demonstrated rapid tissue penetration and high tumor uptake through in vivo imaging. Moreover, Nectin-4 NDC exhibited noteworthy dose-dependent anti-tumor efficacy in in vivo studies.
We have engineered a Nectin-4 NDC with elevated affinity and effective tumor uptake, further establishing its potential as a therapeutic agent for gastric cancer.
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