The identification and optimization of mutations in nanobodies are crucial for enhancing their thera-peutic potential in disease prevention and control.However,this process is often complex and time-consuming,which li...The identification and optimization of mutations in nanobodies are crucial for enhancing their thera-peutic potential in disease prevention and control.However,this process is often complex and time-consuming,which limit its widespread application in practice.In this study,we developed a work-flow,named Evolutionary-Nanobody(EvoNB),to predict key mutation sites of nanobodies by combining protein language models(PLMs)and molecular dynamic(MD)simulations.By fine-tuning the ESM2 model on a large-scale nanobody dataset,the ability of EvoNB to capture specific sequence features of nanobodies was significantly enhanced.The fine-tuned EvoNB model demonstrated higher predictive accuracy in the conserved framework and highly variable complementarity-determining regions of nanobodies.Additionally,we selected four widely representative nanobodyeantigen complexes to verify the predicted effects of mutations.MD simulations analyzed the energy changes caused by these mu-tations to predict their impact on binding affinity to the targets.The results showed that multiple mu-tations screened by EvoNB significantly enhanced the binding affinity between nanobody and its target,further validating the potential of this workflow for designing and optimizing nanobody mutations.Additionally,sequence-based predictions are generally less dependent on structural absence,allowing them to be more easily integrated with tools for structural predictions,such as AlphaFold 3.Through mutation prediction and systematic analysis of key sites,we can quickly predict the most promising variants for experimental validation without relying on traditional evolutionary or selection processes.The EvoNB workflow provides an effective tool for the rapid optimization of nanobodies and facilitates the application of PLMs in the biomedical field.展开更多
Nanobodies have been extensively demonstrated in biomedical imaging and therapy. However, nanobody probes often suffer from rapid renal clearance due to its small size. Herein, we reported a recombinant nanobody with ...Nanobodies have been extensively demonstrated in biomedical imaging and therapy. However, nanobody probes often suffer from rapid renal clearance due to its small size. Herein, we reported a recombinant nanobody with a 200 amino-acid polypeptide chain consisting of Pro, Ala, and Ser (PAS) at the C-terminal, which can be easily expressed in Escherichia coli with a high yield. The PASylated nanobody was functionalized with a fluorescent dye and the cell labeling properties were characterized by flow cytometry and confocal microscopy. In vivo fluorescence imaging indicated that the PASylated nanobody showed comparable blood circulation time, but ∼1.5 times higher tumor targeting ability as compared to the PEGylated nanobody of comparable molecular weight. Our findings demonstrate that nanobody PASylation is a promising approach to produce long-circulating nanobody probes for imaging and therapeutic applications.展开更多
African swine fever virus(ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in ...African swine fever virus(ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds.Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase(nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA(cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7%by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.展开更多
Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA...Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA)has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.However,there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.In the present study,the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.Then,two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.Subsequently,two nanobody-HRP fusion proteins were expressed with HEK293T cells.The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA(bELISA)to detect anti-PRV-gE antibodies.Through optimizing the conditions of bELISA,the amount of coated antigen was 200 ng per well,and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.The cut-off value of bELISA was 24.20%,and the sensitivity and specificity were 96.43 and 92.63%,respectively.By detecting 233 clinical pig sera with the developed bELISA and a commercial kit,the results showed that the coincidence rate of two assays was 93.99%.Additionallly,epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.Simple,great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.The bELISA could be used for monitoring and eradicating PR.展开更多
Objective:We aimed to develop a novel anti-HIF-1αintrabody to decrease gemcitabine resistance in pancreatic cancer patients.Methods:Surface plasmon resonance and glutathione S-transferase pull-down assays were conduc...Objective:We aimed to develop a novel anti-HIF-1αintrabody to decrease gemcitabine resistance in pancreatic cancer patients.Methods:Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1αVHH212[a single-domain antibody(nanobody)].Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α(HIF-1α)and VHH212.The real-time polymerase chain reaction(PCR)and Western blot analyses were performed to identify the expressions of HIF-1αand VEGF-A in pancreatic ductal adenocarcinoma cell lines.The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay.Finally,a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment.Immunohistochemistry analysis was conducted to detect the expressions of HIF-1αand VEGF-A in tumor tissues.Results:VHH212 was stably expressed in tumor cells with low cytotoxicity,high affinity,specific subcellular localization,and neutralization of HIF-1αin the cytoplasm or nucleus.The binding affinity between VHH212 and the HIF-1αPAS-B domain was 42.7 n M.Intrabody competitive inhibition of the HIF-1αheterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro.Compared with single agent gemcitabine,co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44%tumor inhibition.Conclusions:We developed an anti-HIF-1αnanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer.The combination of VHH212 and gemcitabine significantly inhibited tumor development.These results suggested that combined use of anti-HIF-1αnanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.展开更多
Immunosensing methods are biosensing techniques based on specific recognition of an antigen-antibody immunocomplex,which have become commonly used in safeguarding public health.Taking advantage of antibody-related bio...Immunosensing methods are biosensing techniques based on specific recognition of an antigen-antibody immunocomplex,which have become commonly used in safeguarding public health.Taking advantage of antibody-related biotechnological advances,the utilization of an antigen-binding fragment of a heavy-chain-only antibody termed as'nanobody'holds significant biomedical potential.Compared with the conventional full-length antibody,a single-domain nanobody retaining cognate antigen specificity possesses remarkable physicochemical stability and structural adaptability,which enables a flexible and efficient molecular design of the immunosensing strategy.This minireview aims to summarize the recent progress in immunosensing methods using nanobody targeting tumor markers,environmental pollutants,and foodborne microbes.展开更多
Microcystis species identification is essential for ecological studies and water bloom control.Immunoassays are more specific and convenient and several approaches have been used to develop for diagnosing harmful red ...Microcystis species identification is essential for ecological studies and water bloom control.Immunoassays are more specific and convenient and several approaches have been used to develop for diagnosing harmful red tide algae.Howeve r,inve stigations onMicrocystis identification using immunological approaches are still in the initial stage.In this study.Microcystis aeruginosa PCC7806 lysates were utilized as coated antigens to enrich and screen specific Microcystis nanobodies from a human domain antibody display library.After three rounds of enrichment,10 positive monoclonal particles were isolated from the library and the most two positive nanobodies(DAb2 and Dab3)were effectively produced in Escherichia coli BL21.Finally,the DAb2 showed specific immune binding to different Microcystis by the immuno-dot blot assay.This antibody could be used to establish an immunological method to identify Microcystis.展开更多
Highly efficient removal of tumor necrosis factor-α(TNF-α)from plasma by hemoperfusion for autoim-mune disease therapy remains a challenge in the clinical field owing to the low adsorption capacity and poor blood co...Highly efficient removal of tumor necrosis factor-α(TNF-α)from plasma by hemoperfusion for autoim-mune disease therapy remains a challenge in the clinical field owing to the low adsorption capacity and poor blood compatibility of adsorbents.In this work,a new class of nanobody(Nb)-coupled antifouling polyvinyl alcohol(PVA)beads was constructed as an immunosorbent for the selective removal of TNF-αfrom plasma.Notably,our immunosorbent exhibited an exceptionally high specific TNF-αadsorption ca-pacity of 416.9 ng/g in human plasma(at a plasma-to-adsorbent ratio of 300).More importantly,the ob-tained adsorbent beads showed outstanding blood compatibility.In addition,during in vivo experiments,the blood circulation device was constructed to remove TNF-αin rat models,proving that the beads had good removal performance(∼85%/60 min).Furthermore,95%of the original capacity was retained after 6-month storage,showed strong stability and prolonged storage of PVA-Nb.Above all,the results indi-cate that the novel PVA-Nb immunosorbent has possible clinical applications for treating autoimmune diseases in the clinic.展开更多
To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and q...To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and quantification.However,it requires expensive instrumentation and is not appropriate for bedside use.Using soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human samples.Under optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and accuracy.The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026.In addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency.The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds.These methods could be easily implemented by the bedside,in the field in remote areas or in veterinary practice.This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy.Attributes of nanobody based pharmaceutical assays are discussed.展开更多
Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhI...Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNa2b.RhIFNa2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNa2b detection assay had a limit of detection of 1 mg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.展开更多
Nanobody is the variable region of heavy chain antibody, with obvious characteristics such as small size, good stability and solubility. Many useful nanobodies have been isolated by
The sudden emergence of severe acute respiratory syndrome coronavirus(SARS-CoV) has caused global panic in 2003,and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is availab...The sudden emergence of severe acute respiratory syndrome coronavirus(SARS-CoV) has caused global panic in 2003,and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available;thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain(RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensinconverting enzyme 2(ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.展开更多
Porcine reproductive and respiratory syndrome virus(PRRSV)infection causes significant economic loss to the global pig industry.Genotype 1 and 2 PRRSV(PRRSV-1 and-2)infections have been reported in China,Europe and Am...Porcine reproductive and respiratory syndrome virus(PRRSV)infection causes significant economic loss to the global pig industry.Genotype 1 and 2 PRRSV(PRRSV-1 and-2)infections have been reported in China,Europe and America.For accurate prevention,nanobodies were first used as diagnostic reagents for PRRSV typing.In this study three nanobodies targeting both PRRSV-1 and-2,two targeting PRRSV-1 and three targeting PRRSV-2,were screened and produced.To develop two competitive ELISAs(cELISAs),the g1-2-PRRSV-Nb3-HRP nanobody was chosen for the g1-2-cELISA,to detect common antibodies against PRRSV-1 and-2,and the g1-PRRSV-Nb136-HRP nanobody was chosen for the g1-cELISA,to detect anti-PRRSV-1 antibodies.The two cELISAs were developed using PRRSV-1-N protein as coating antigen,and the amounts for both were 100 ng/well.The optimized dilution of testing pig sera was 1:20,the optimized reaction times were 30 min,and the colorimetric reaction times were 15 min.Then,the cut-off values of the g1-2-cELISA and g1-cELISA were 26.6%and 35.6%,respectively.Both of them have high sensitivity,strong specificity,good repeatability,and stability.In addition,for the 1534 clinical pig sera,an agreement rate of 99.02%(Kappa values=0.97)was determined between the g1-2-cELISA and the commercial IDEXX ELISA kit.For the g1-cELSIA,it can specifically detect anti-PRRSV-1 antibodies in the clinical pig sera.Importantly,combining two nanobody-based cELISAs can differentially detect antibodies against PRRSV-1 and-2.展开更多
Whenever there is no adequate DNA replication <em>in vitro</em>, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are c...Whenever there is no adequate DNA replication <em>in vitro</em>, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are called vectors or cloning vehicles. These days, there is a high demand to manufacture recombinant and nanobodies which are required in biomedical research and for therapeutic and diagnostic purposes. In order to do so, <em>E. coli</em>, insect cells, or mammalian cells have been used to express and purify protein for nanobody production. This paper explains the experimental trials on the cloning of the Nanobody cDNA mock-up in pHEN6c plasmid vector from the subcultured <em>E. coli</em> sample taken from the lab. The concentration and purity of DNA plasmid were evaluated by UV spectrophotometer and agarose gel electrophoresis following plasmid DNA Purification from <em>E. coli</em> by alkaline lysis. Based on this, the concentration of the isolated pHEN6c plasmid DNA was found 44.7 ng/μl or 0.0447 μg/μl. Whereas, the purity at absorbance (A260/A280) was 0.893/0.501 = 1.78. Moreover, its yield was 2.235 μg. In addition, its transformation efficiency was 21.68 μg/μl. On the other hand, the molecular weight of the Nanobody and vector were 569 and 2717 respectively. Generally, most of the protocols used to clone a fragment of DNA, might not work very well with PCR products.展开更多
Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dyn...Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (M<sup>pro</sup>) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M<sup>pro</sup>: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.展开更多
The cardiac microenvironment profoundly restricts the efficacy of myocardial regeneration tactics for the treatment of myocardial infarction(MI).A prospective approach for MI therapeutics encompasses the combined stra...The cardiac microenvironment profoundly restricts the efficacy of myocardial regeneration tactics for the treatment of myocardial infarction(MI).A prospective approach for MI therapeutics encompasses the combined strategy of scavenging reactive oxygen species(ROS)to alleviate oxidative stress injury and facilitating macrophage polarization towards the regenerative M2 phenotype.In this investigation,we fabricated a ROSsensitive hydrogel engineered to deliver our previously engineered IL-1β-VHH for myocardial restoration.In mouse and rat models of myocardial infarction,the therapeutic gel was injected into the pericardial cavity,effectively disseminated over the heart surface,forming an in situ epicardial patch.The IL-1β-VHH released from the hydrogel exhibited penetrative potential into the myocardium.Our results imply that this infarct-targeting gel can adhere to the damaged cardiac tissue and augment the quantity of anti-IL-1βantibodies.Moreover,the anti-IL-1βhydrogel safeguards cardiomyocytes from apoptosis by neutralizing IL-1βand inducing M2-type polarization within the myocardial infarction regions,thereby facilitating therapeutic cardiac repair.Our results emphasize the effectiveness of this synergistic comprehensive treatment modality in the management of MI and showcase its considerable potential for promoting recovery in infarcted hearts.展开更多
Glioma represents the most prevalent malignant tumor of the central nervous system,with chemotherapy serving as an essential adjunctive treatment.However,most chemotherapeutic agents exhibit limited ability to penetra...Glioma represents the most prevalent malignant tumor of the central nervous system,with chemotherapy serving as an essential adjunctive treatment.However,most chemotherapeutic agents exhibit limited ability to penetrate the blood-brain barrier(BBB).This study introduced a novel dualtargeting strategy for glioma therapy by modulating the formation of nanobody-driven protein coronas to enhance the brain and tumor-targeting efficiency of hydrophobic cisplatin prodrug-loaded lipid nanoparticles(C8Pt-Ls).Specifically,nanobodies(Nbs)with fibrinogen-binding capabilities were conjugated to the surface of C8Pt-Ls,resulting in the generation of Nb-C8Pt-Ls.Within the bloodstream,Nb-C8Pt-Ls could bound more fibrinogen,forming the protein corona that specifically interacted with LRP-1,a receptor highly expressed on the BBB.This interaction enabled a“Hitchhiking Effect”mechanism,facilitating efficient trans-BBB transport and promoting effective brain targeting.Additionally,the protein corona interacted with LRP-1,which is also overexpressed in glioma cells,achieving precise tumor targeting.Computational simulations and SPR detection clarified the molecular interaction mechanism of the Nb-fibrinogen-(LRP-1)complex,confirming its binding specificity and stability.Our results demonstrated that this strategy significantly enhanced C8Pt accumulation in brain tissues and tumors,induced apoptosis in glioma cells,and improved therapeutic efficacy.This study provides a novel framework for glioma therapy and underscores the potential of protein corona modulation-based dual-targeting strategies in advancing treatments for brain tumors.展开更多
This study presents a novel approach targeting CD155,an overexpressed protein in lung adenocarcinoma(LUAD),using nanobodies with exceptional precision and efficacy.The significant upregulation of CD155 in LUAD,associa...This study presents a novel approach targeting CD155,an overexpressed protein in lung adenocarcinoma(LUAD),using nanobodies with exceptional precision and efficacy.The significant upregulation of CD155 in LUAD,associated with poor patient outcomes,highlights its potential as a therapeutic target.An anti-CD155 nanobody(A5 Nb)is developed that binds to CD155-positive lung cancer cells with high affinity(A5 Nb K_(d)=0.23 nM).The complementarity-determining region of A5 Nb forms hydrophobic interactions and hydrogen bonds with CD155,promoting selective binding and stabilization of A5 Nb-CD155 complex.展开更多
Epitope tags are widely used for detecting,modifying,or purifying proteins of interest,but their range of application is often limited.Recently,the rationally designed ALFA tag and its ALFA nanobody have expanded the ...Epitope tags are widely used for detecting,modifying,or purifying proteins of interest,but their range of application is often limited.Recently,the rationally designed ALFA tag and its ALFA nanobody have expanded the repertoire of epitope tags and emerged as a highly versatile system characterized in various animal models,outperforming existing tags.Here,we evaluated the ALFA tag/ALFA nanobody technology in plants and demonstrated its application for in planta protein detection across multiple compartments and cellular structures,protein-protein interaction studies,protein immunoprecipitation,inducedproximity approaches,and super-resolution microscopy.Most importantly,we highlight the potential of the ALFA tagging technology for proteins that are difficult to tag due to topological or functional constraints.We provide proof of concept for the ALFA tag technology in the detection and functional analysis of the Arabidopsis IRT1 Fe transporter.Overall,this versatile and validated toolbox of ALFA tag and ALFA nanobody applications will serve as a valuable resource for functional studies in plants.展开更多
Neutralizing antibodies have been designed to specifically target and bind to the receptor binding domain(RBD)of spike(S)protein to block severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus from attaching...Neutralizing antibodies have been designed to specifically target and bind to the receptor binding domain(RBD)of spike(S)protein to block severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus from attaching to angiotensin converting enzyme 2(ACE2).This study reports a distinctive nanobody,designated as VHH21,that directly catalyzes the S-trimer into an irreversible transition state through postfusion conformational changes.Derived from camels immunized with multiple antigens,a set of nanobodies with high affinity for the S1 protein displays abilities to neutralize pseudovirion infections with a broad resistance to variants of concern of SARS-CoV-2,including SARS-CoV and BatRaTG13.Importantly,a super-resolution screening and analysis platform based on visual fluorescence probes was designed and applied to monitor single proteins and protein subunits.A spontaneously occurring dimeric form of VHH21 was obtained to rapidly destroy the S-trimer.Structural analysis via cryogenic electron microscopy revealed that VHH21 targets specific conserved epitopes on the S protein,distinct from the ACE2 binding site on the RBD,which destabilizes the fusion process.This research highlights the potential of VHH21 as an abzyme-like nanobody(nanoabzyme)possessing broad-spectrum binding capabilities and highly effective anti-viral properties and offers a promising strategy for combating coronavirus outbreaks.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.:92477103,22273023,12474285 and 22373116)the National Key R&D Program of China(Grant No.:2019YFA0905200)+5 种基金Shanghai Municipal Natural Science Foundation(Grant No.:23ZR1418200)Natural Science Foundation of Chongqing,China(Grant No.:CSTB2023NSCQ-MSX0616)Shanghai Frontiers Science Center of Molecule Intelligent SynthesesShanghai Future Discipline Program(Quantum Science and Tech-nology)Shanghai Municipal Education Commission’s“Artificial Intelligence-Driven Research Paradigm Reform and Discipline Advancement Program”the Fundamental Research Funds for the Central Universities.
文摘The identification and optimization of mutations in nanobodies are crucial for enhancing their thera-peutic potential in disease prevention and control.However,this process is often complex and time-consuming,which limit its widespread application in practice.In this study,we developed a work-flow,named Evolutionary-Nanobody(EvoNB),to predict key mutation sites of nanobodies by combining protein language models(PLMs)and molecular dynamic(MD)simulations.By fine-tuning the ESM2 model on a large-scale nanobody dataset,the ability of EvoNB to capture specific sequence features of nanobodies was significantly enhanced.The fine-tuned EvoNB model demonstrated higher predictive accuracy in the conserved framework and highly variable complementarity-determining regions of nanobodies.Additionally,we selected four widely representative nanobodyeantigen complexes to verify the predicted effects of mutations.MD simulations analyzed the energy changes caused by these mu-tations to predict their impact on binding affinity to the targets.The results showed that multiple mu-tations screened by EvoNB significantly enhanced the binding affinity between nanobody and its target,further validating the potential of this workflow for designing and optimizing nanobody mutations.Additionally,sequence-based predictions are generally less dependent on structural absence,allowing them to be more easily integrated with tools for structural predictions,such as AlphaFold 3.Through mutation prediction and systematic analysis of key sites,we can quickly predict the most promising variants for experimental validation without relying on traditional evolutionary or selection processes.The EvoNB workflow provides an effective tool for the rapid optimization of nanobodies and facilitates the application of PLMs in the biomedical field.
基金support by the National Key R&D Program of China(Grant No.2020YFA0909000)National Natural Science Foundation of China(Grant Nos.62235007 and 22204070)+2 种基金Shenzhen Science and Technology Program(Grant Nos.KQTD20170810111314625,JCYJ20210324115807021,and SGDX20211123114002003)Shenzhen Bay Laboratory(SZBL 2021080601002)Guangdong Provincial Key Laboratory of Advanced Biomaterials(2022 B1212010003).
文摘Nanobodies have been extensively demonstrated in biomedical imaging and therapy. However, nanobody probes often suffer from rapid renal clearance due to its small size. Herein, we reported a recombinant nanobody with a 200 amino-acid polypeptide chain consisting of Pro, Ala, and Ser (PAS) at the C-terminal, which can be easily expressed in Escherichia coli with a high yield. The PASylated nanobody was functionalized with a fluorescent dye and the cell labeling properties were characterized by flow cytometry and confocal microscopy. In vivo fluorescence imaging indicated that the PASylated nanobody showed comparable blood circulation time, but ∼1.5 times higher tumor targeting ability as compared to the PEGylated nanobody of comparable molecular weight. Our findings demonstrate that nanobody PASylation is a promising approach to produce long-circulating nanobody probes for imaging and therapeutic applications.
基金supported by the Natural Science Foundation of China (grant no. 31941016 and 31972676)the Natural Science Foundation of Shaanxi Province of China (grant no. 2022JC-12)the Key R&D Program of Shaanxi Province (grant no. S2022-YF-YBNY0673)
文摘African swine fever virus(ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds.Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase(nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA(cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7%by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.
基金supported by the National Natural Science Foundation of China(32273041)the Key R&D Program of Shaanxi Province,China(2022NY-104)the Natural Science Foundation of Shaanxi Province,China(2022JC-12)。
文摘Pseudorabies(PR)is an acute infectious disease of pigs caused by the PR virus(PRV)and results in great economic losses to the pig industry worldwide.PRV glycoprotein E(gE)-based enzyme-linked immunosorbent assay(ELISA)has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.However,there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.In the present study,the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.Then,two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.Subsequently,two nanobody-HRP fusion proteins were expressed with HEK293T cells.The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA(bELISA)to detect anti-PRV-gE antibodies.Through optimizing the conditions of bELISA,the amount of coated antigen was 200 ng per well,and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.The cut-off value of bELISA was 24.20%,and the sensitivity and specificity were 96.43 and 92.63%,respectively.By detecting 233 clinical pig sera with the developed bELISA and a commercial kit,the results showed that the coincidence rate of two assays was 93.99%.Additionallly,epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.Simple,great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.The bELISA could be used for monitoring and eradicating PR.
基金supported by grants from the National Key Research and Development Project(Grant No.2019YFA0905600)the Major State Basic Research Development Program of the Natural Science Foundation of Shandong Province in China(Grant No.ZR2020ZD11)+2 种基金the National Natural Science Foundation of China(Grant Nos.81772633 and 31470967)the Science and Technology Program of Tianjin,China(Grant No.19YFSLQY00110)the Taishan Scholars Program of Shandong Province。
文摘Objective:We aimed to develop a novel anti-HIF-1αintrabody to decrease gemcitabine resistance in pancreatic cancer patients.Methods:Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1αVHH212[a single-domain antibody(nanobody)].Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α(HIF-1α)and VHH212.The real-time polymerase chain reaction(PCR)and Western blot analyses were performed to identify the expressions of HIF-1αand VEGF-A in pancreatic ductal adenocarcinoma cell lines.The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay.Finally,a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment.Immunohistochemistry analysis was conducted to detect the expressions of HIF-1αand VEGF-A in tumor tissues.Results:VHH212 was stably expressed in tumor cells with low cytotoxicity,high affinity,specific subcellular localization,and neutralization of HIF-1αin the cytoplasm or nucleus.The binding affinity between VHH212 and the HIF-1αPAS-B domain was 42.7 n M.Intrabody competitive inhibition of the HIF-1αheterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro.Compared with single agent gemcitabine,co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44%tumor inhibition.Conclusions:We developed an anti-HIF-1αnanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer.The combination of VHH212 and gemcitabine significantly inhibited tumor development.These results suggested that combined use of anti-HIF-1αnanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.
基金the National Natural Science Foundation of China(No.U1703118)Natural Science Foundation of Jiangsu Higher Education Institutions of China(No.19KJA310003)Natural Science Foundation of Jiangsu Province(No.BK20181364)。
文摘Immunosensing methods are biosensing techniques based on specific recognition of an antigen-antibody immunocomplex,which have become commonly used in safeguarding public health.Taking advantage of antibody-related biotechnological advances,the utilization of an antigen-binding fragment of a heavy-chain-only antibody termed as'nanobody'holds significant biomedical potential.Compared with the conventional full-length antibody,a single-domain nanobody retaining cognate antigen specificity possesses remarkable physicochemical stability and structural adaptability,which enables a flexible and efficient molecular design of the immunosensing strategy.This minireview aims to summarize the recent progress in immunosensing methods using nanobody targeting tumor markers,environmental pollutants,and foodborne microbes.
基金Supported by the National Natural Science Foundation of China(Nos.31971561,31370217,31701724)the Research Program of Jiangsu Province of China。
文摘Microcystis species identification is essential for ecological studies and water bloom control.Immunoassays are more specific and convenient and several approaches have been used to develop for diagnosing harmful red tide algae.Howeve r,inve stigations onMicrocystis identification using immunological approaches are still in the initial stage.In this study.Microcystis aeruginosa PCC7806 lysates were utilized as coated antigens to enrich and screen specific Microcystis nanobodies from a human domain antibody display library.After three rounds of enrichment,10 positive monoclonal particles were isolated from the library and the most two positive nanobodies(DAb2 and Dab3)were effectively produced in Escherichia coli BL21.Finally,the DAb2 showed specific immune binding to different Microcystis by the immuno-dot blot assay.This antibody could be used to establish an immunological method to identify Microcystis.
基金supported by grants from the National Key R&D Program of China(No.2017YFC1104401)the National Natu-ral Science Foundation of China(No.81771986)+1 种基金the Natural Sci-ence Foundation of Tianjin(No.18YFZCSY00860)the Scientific Research Translational Foundation of Wenzhou Safety(Emergency)Institute of Tianjin University(No.TJUWYY2022004).
文摘Highly efficient removal of tumor necrosis factor-α(TNF-α)from plasma by hemoperfusion for autoim-mune disease therapy remains a challenge in the clinical field owing to the low adsorption capacity and poor blood compatibility of adsorbents.In this work,a new class of nanobody(Nb)-coupled antifouling polyvinyl alcohol(PVA)beads was constructed as an immunosorbent for the selective removal of TNF-αfrom plasma.Notably,our immunosorbent exhibited an exceptionally high specific TNF-αadsorption ca-pacity of 416.9 ng/g in human plasma(at a plasma-to-adsorbent ratio of 300).More importantly,the ob-tained adsorbent beads showed outstanding blood compatibility.In addition,during in vivo experiments,the blood circulation device was constructed to remove TNF-αin rat models,proving that the beads had good removal performance(∼85%/60 min).Furthermore,95%of the original capacity was retained after 6-month storage,showed strong stability and prolonged storage of PVA-Nb.Above all,the results indi-cate that the novel PVA-Nb immunosorbent has possible clinical applications for treating autoimmune diseases in the clinic.
基金supported by NIEHS(RIVER Award,R35 ES030443)NIEHS(Superfund Award,P42 ES004699)+6 种基金NINDS(Counter ActProgram U54 NS127758)Juvenile Diabetes Research Foundation(2-SRA-2022-1210-S-B)Guangzhou Science and Technology Foundation(Grant No.:201903010034)Natural Resources Science Foundation of Guangdong Province(Grant No.:2018A030313926)Science and Technology Foundation Key R&D Program of Guangdong Province(Grant Nos.:2019B020209009 and 2019B020218009)R&D Program of Guangdong Province Drug Administration(Grant Nos.:2021TDZ09 and 2021YDZ06)supported by China Scholarship Council(CSC)(202108440382).
文摘To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and quantification.However,it requires expensive instrumentation and is not appropriate for bedside use.Using soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human samples.Under optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and accuracy.The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026.In addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency.The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds.These methods could be easily implemented by the bedside,in the field in remote areas or in veterinary practice.This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy.Attributes of nanobody based pharmaceutical assays are discussed.
基金support was provided by the National Science and Technology Major Project(Grant No.:2015ZX09501008)。
文摘Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNa2b.RhIFNa2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNa2b detection assay had a limit of detection of 1 mg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.
文摘Nanobody is the variable region of heavy chain antibody, with obvious characteristics such as small size, good stability and solubility. Many useful nanobodies have been isolated by
基金This study was supported by SKLPBS1805 and 2019-JCJQ-JJ-167(to G.Z.)supported by the National Science Fund for Distinguished Young Scholar(No.81925025)+1 种基金the Innovative Research Group(No.81621005)from the NSFCthe Innovation Fund for Medical Sciences(No.2019-I2M-5-049)from the Chinese Academy of Medical Sciences。
文摘The sudden emergence of severe acute respiratory syndrome coronavirus(SARS-CoV) has caused global panic in 2003,and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available;thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain(RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensinconverting enzyme 2(ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.
基金funded by grants from the National Key R&D Program of China(2023YFD1800304)the National Natural Science Foundation of China to QZ(32273041)+1 种基金the Natural Science Foundation of Shaanxi Province of China(2022JC-12)the Central Public-interest Scientific Institution Basal Research Fund,National Data Center of Animal Health.
文摘Porcine reproductive and respiratory syndrome virus(PRRSV)infection causes significant economic loss to the global pig industry.Genotype 1 and 2 PRRSV(PRRSV-1 and-2)infections have been reported in China,Europe and America.For accurate prevention,nanobodies were first used as diagnostic reagents for PRRSV typing.In this study three nanobodies targeting both PRRSV-1 and-2,two targeting PRRSV-1 and three targeting PRRSV-2,were screened and produced.To develop two competitive ELISAs(cELISAs),the g1-2-PRRSV-Nb3-HRP nanobody was chosen for the g1-2-cELISA,to detect common antibodies against PRRSV-1 and-2,and the g1-PRRSV-Nb136-HRP nanobody was chosen for the g1-cELISA,to detect anti-PRRSV-1 antibodies.The two cELISAs were developed using PRRSV-1-N protein as coating antigen,and the amounts for both were 100 ng/well.The optimized dilution of testing pig sera was 1:20,the optimized reaction times were 30 min,and the colorimetric reaction times were 15 min.Then,the cut-off values of the g1-2-cELISA and g1-cELISA were 26.6%and 35.6%,respectively.Both of them have high sensitivity,strong specificity,good repeatability,and stability.In addition,for the 1534 clinical pig sera,an agreement rate of 99.02%(Kappa values=0.97)was determined between the g1-2-cELISA and the commercial IDEXX ELISA kit.For the g1-cELSIA,it can specifically detect anti-PRRSV-1 antibodies in the clinical pig sera.Importantly,combining two nanobody-based cELISAs can differentially detect antibodies against PRRSV-1 and-2.
文摘Whenever there is no adequate DNA replication <em>in vitro</em>, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are called vectors or cloning vehicles. These days, there is a high demand to manufacture recombinant and nanobodies which are required in biomedical research and for therapeutic and diagnostic purposes. In order to do so, <em>E. coli</em>, insect cells, or mammalian cells have been used to express and purify protein for nanobody production. This paper explains the experimental trials on the cloning of the Nanobody cDNA mock-up in pHEN6c plasmid vector from the subcultured <em>E. coli</em> sample taken from the lab. The concentration and purity of DNA plasmid were evaluated by UV spectrophotometer and agarose gel electrophoresis following plasmid DNA Purification from <em>E. coli</em> by alkaline lysis. Based on this, the concentration of the isolated pHEN6c plasmid DNA was found 44.7 ng/μl or 0.0447 μg/μl. Whereas, the purity at absorbance (A260/A280) was 0.893/0.501 = 1.78. Moreover, its yield was 2.235 μg. In addition, its transformation efficiency was 21.68 μg/μl. On the other hand, the molecular weight of the Nanobody and vector were 569 and 2717 respectively. Generally, most of the protocols used to clone a fragment of DNA, might not work very well with PCR products.
文摘Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (M<sup>pro</sup>) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M<sup>pro</sup>: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.
基金funded by The National Natural Science Foundation of China(NSFC)(82170256)Guangdong Major Project of Basic and Applied Basic Research(2023B0303000005)+3 种基金The National Key Research and Development Program of China(2018YFA0108700)Guangdong Provincial Special Support Program for Prominent Talents(2021JC06Y656)Science and Technology Planning Project of Guangdong Province(2022B1212010010)the Special Project of Dengfeng Program of Guangdong Provincial People’s Hospital(KJ012019119).
文摘The cardiac microenvironment profoundly restricts the efficacy of myocardial regeneration tactics for the treatment of myocardial infarction(MI).A prospective approach for MI therapeutics encompasses the combined strategy of scavenging reactive oxygen species(ROS)to alleviate oxidative stress injury and facilitating macrophage polarization towards the regenerative M2 phenotype.In this investigation,we fabricated a ROSsensitive hydrogel engineered to deliver our previously engineered IL-1β-VHH for myocardial restoration.In mouse and rat models of myocardial infarction,the therapeutic gel was injected into the pericardial cavity,effectively disseminated over the heart surface,forming an in situ epicardial patch.The IL-1β-VHH released from the hydrogel exhibited penetrative potential into the myocardium.Our results imply that this infarct-targeting gel can adhere to the damaged cardiac tissue and augment the quantity of anti-IL-1βantibodies.Moreover,the anti-IL-1βhydrogel safeguards cardiomyocytes from apoptosis by neutralizing IL-1βand inducing M2-type polarization within the myocardial infarction regions,thereby facilitating therapeutic cardiac repair.Our results emphasize the effectiveness of this synergistic comprehensive treatment modality in the management of MI and showcase its considerable potential for promoting recovery in infarcted hearts.
基金supported by the National Natural Science Foundation of China(22107080 and 82300113).
文摘Glioma represents the most prevalent malignant tumor of the central nervous system,with chemotherapy serving as an essential adjunctive treatment.However,most chemotherapeutic agents exhibit limited ability to penetrate the blood-brain barrier(BBB).This study introduced a novel dualtargeting strategy for glioma therapy by modulating the formation of nanobody-driven protein coronas to enhance the brain and tumor-targeting efficiency of hydrophobic cisplatin prodrug-loaded lipid nanoparticles(C8Pt-Ls).Specifically,nanobodies(Nbs)with fibrinogen-binding capabilities were conjugated to the surface of C8Pt-Ls,resulting in the generation of Nb-C8Pt-Ls.Within the bloodstream,Nb-C8Pt-Ls could bound more fibrinogen,forming the protein corona that specifically interacted with LRP-1,a receptor highly expressed on the BBB.This interaction enabled a“Hitchhiking Effect”mechanism,facilitating efficient trans-BBB transport and promoting effective brain targeting.Additionally,the protein corona interacted with LRP-1,which is also overexpressed in glioma cells,achieving precise tumor targeting.Computational simulations and SPR detection clarified the molecular interaction mechanism of the Nb-fibrinogen-(LRP-1)complex,confirming its binding specificity and stability.Our results demonstrated that this strategy significantly enhanced C8Pt accumulation in brain tissues and tumors,induced apoptosis in glioma cells,and improved therapeutic efficacy.This study provides a novel framework for glioma therapy and underscores the potential of protein corona modulation-based dual-targeting strategies in advancing treatments for brain tumors.
基金supported by NRF and NST grants funded by Korea government(MSIT)(2021M3H4A1A02051048,2023R1A2C2005185,RS-2024-00348576,RS-2024-00438316,RS-2024-00459749,2024-00338524,RS-2024-00338397)KEIT grants funded by Korea government(MOTIE)(RS-2022-00154853,RS-2024-00403563,RS-2024-00432382)+2 种基金KEITI grant funded by Korea government(ME)(2021003370003)IPET grant funded by Korea government(MAFRA)(RS-2024-00401639)Nanomedical Devices Development Program of National Nano Fab Center,and KRIBB Research Initiative Program(KGM1322511 and KGM1032511).
文摘This study presents a novel approach targeting CD155,an overexpressed protein in lung adenocarcinoma(LUAD),using nanobodies with exceptional precision and efficacy.The significant upregulation of CD155 in LUAD,associated with poor patient outcomes,highlights its potential as a therapeutic target.An anti-CD155 nanobody(A5 Nb)is developed that binds to CD155-positive lung cancer cells with high affinity(A5 Nb K_(d)=0.23 nM).The complementarity-determining region of A5 Nb forms hydrophobic interactions and hydrogen bonds with CD155,promoting selective binding and stabilization of A5 Nb-CD155 complex.
基金supported by research grants from the French National Research Agency(grant nos.ANR-21-CE20-0046 to G.V.and 22-PESV0002 to J.N.)the French Laboratory of Excellence(project“TULIP”grant nos.ANR-10-LABX-41 and ANR-11-IDEX-0002-02 to G.V.).
文摘Epitope tags are widely used for detecting,modifying,or purifying proteins of interest,but their range of application is often limited.Recently,the rationally designed ALFA tag and its ALFA nanobody have expanded the repertoire of epitope tags and emerged as a highly versatile system characterized in various animal models,outperforming existing tags.Here,we evaluated the ALFA tag/ALFA nanobody technology in plants and demonstrated its application for in planta protein detection across multiple compartments and cellular structures,protein-protein interaction studies,protein immunoprecipitation,inducedproximity approaches,and super-resolution microscopy.Most importantly,we highlight the potential of the ALFA tagging technology for proteins that are difficult to tag due to topological or functional constraints.We provide proof of concept for the ALFA tag technology in the detection and functional analysis of the Arabidopsis IRT1 Fe transporter.Overall,this versatile and validated toolbox of ALFA tag and ALFA nanobody applications will serve as a valuable resource for functional studies in plants.
基金supported by grants from National Natural Science Foundation of China(Nos.92374206,82225037,31900871,and 32241027)National Key R&D Program of China(Nos.2022YFA1303602 and 2021YFA1301402)+4 种基金the Strategic Priority Research Program of CAS(Nos.XDB37030206,XDB29040102,XDB37040101,and XDB29030103)Shanghai Municipal Science and Technology Major Project(No.ZD2021CY001)Basic Research Program Based on Major Scientific Infrastructures of CAS(No.JZHKYPT-2021-05)Key Laboratory of Biomacromolecules of CAS(No.ZGD-2023-05)Beijing Nova Program(No.20230484239 to Duanfang Cao).
文摘Neutralizing antibodies have been designed to specifically target and bind to the receptor binding domain(RBD)of spike(S)protein to block severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus from attaching to angiotensin converting enzyme 2(ACE2).This study reports a distinctive nanobody,designated as VHH21,that directly catalyzes the S-trimer into an irreversible transition state through postfusion conformational changes.Derived from camels immunized with multiple antigens,a set of nanobodies with high affinity for the S1 protein displays abilities to neutralize pseudovirion infections with a broad resistance to variants of concern of SARS-CoV-2,including SARS-CoV and BatRaTG13.Importantly,a super-resolution screening and analysis platform based on visual fluorescence probes was designed and applied to monitor single proteins and protein subunits.A spontaneously occurring dimeric form of VHH21 was obtained to rapidly destroy the S-trimer.Structural analysis via cryogenic electron microscopy revealed that VHH21 targets specific conserved epitopes on the S protein,distinct from the ACE2 binding site on the RBD,which destabilizes the fusion process.This research highlights the potential of VHH21 as an abzyme-like nanobody(nanoabzyme)possessing broad-spectrum binding capabilities and highly effective anti-viral properties and offers a promising strategy for combating coronavirus outbreaks.
