Development of a radiolabeled site-specific single-domain antibody positron emission tomography probe for monitoring PD-L1 expression in cancer

Despite advances in immunotherapy for the treatment of cancers,not all patients can benefit from programmed cell death ligand 1(PD-L1)immune checkpoint blockade therapy.Anti-PD-L1 therapeutic effects reportedly correlate with the PD-L1 expression level;hence,accurate detection of PD-L1 expression can guide immunotherapy to achieve better therapeutic effects.Therefore,based on the high affinity antibody Nb109,a new site-specifically radiolabeled tracer,^(68)Ga-NODA-cysteine,aspartic acid,and valine(CDV)-Nb109,was designed and synthesized to accurately monitor PD-L1 expression.The tracer ^(68)Ga-NODA-CDV-Nb109 was obtained using a site-specific conjugation strategy with a radiochemical yield of about 95%and radiochemical purity of 97%.It showed high affinity for PD-L1 with a dissociation constant of 12.34±1.65 nM.Both the cell uptake assay and positron emission tomography(PET)imaging revealed higher tracer uptake in PD-L1-positive A375-hPD-L1 and U87 tumor cells than in PD-L1-negative A375 tumor cells.Meanwhile,dynamic PET imaging of a NCI-H1299 xenograft indicated that doxorubicin could upregulate PD-L1 expression,allowing timely interventional immunotherapy.In conclusion,this tracer could sensitively and dynamically monitor changes in PD-L1 expression levels in different cancers and help screen patients who can benefit from anti-PD-L1 immunotherapy.

Isolation and optimization of camelid single-domain antibodies:Dirk Saerens'work on nanobodies

It is well established that all camelids have unique antibodies circulating in their blood.Unlike antibodies from all other species,these special antibodies are devoid of light chains,and are composed of a heavy chain homodimer.These so-called heavy-chain antibodies(HCAbs)are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol.These HCAbs are easily purified from serum,and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies.The antigen binding site of the dromedary HCAb comprises one single domain,referred to as VHH or nanobody(Nb),therefore,a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens.The monoclonal Nb is produced well in bacteria,is very stable and highly soluble,and it binds the antigen with high affinity and specificity.Currently,the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors,to diagnose infections,or to treat diseases such as cancer or trypanosomiasis.

Construction of human Fab library and screening of a single-domain antibody of amyloid-beta 42 oligomers

Screening humanized antibodies from a human Fab phage display library is an effective and quick method to obtain beta-amyloid oligomers. Thus, the present study prepared amyloid-beta 42 oli- gomers and constructed a have human Fab phage display library based on blood samples from six healthy people. After three rounds of biopanning in vitro, a human single-domain antibody that spe- cifically recognized amyloid-beta 42 oligomers was identified. Western blot and enzyme-linked im- munosorbent assay demonstrated this antibody bound specifically to human amyloid-beta 42 tetramer and nonamer, but not the monomer or high molecular weight oligomers. This study suc- cessfully constructed a human phage display library and screened a single-domain antibody that specifically recognized amyloid-beta 42 oligomers.

Magnetic Properties for the Single-domain Co Fe_2O_4 Nanoparticles Synthesized by the Hydrothermal Method

The aim of this work was to investigate the size-related magnetism for the single-domain Co Fe2O4 nano-particles synthesized using the hydrothermal method. The effects of the reaction temperature and the reaction time on the lattice constants, particle morphologies, and the room-temperature magnetic properties were studied from the X-ray diffraction, the transmission electron microscope, and the vibrating-sample magnetometer. The experimental results show that the samples are composed of Co Fe2O4 nano-particles with an average crystallite size（D） smaller than 40 nm, and the magnetic properties of the samples can be manipulated in a wide range： the MS values vary from smaller than 50 emu/g to close to 80 emu/g, and the HC values are between about 200 Oe and 2000 Oe. Additionally, the relationship between HC and 1/D^3/2 satisfi es linearship, showing the characteristic of single-domain structure. These results indicate that the single-domain Co Fe2O4 nano-particles with size controlled between the superparamagnetic critical size and single-domain critical size can be easily prepared using this hydrothermal method.

Single-domain antibodies as therapeutics for solid tumor treatment

Single-domain antibodies(sdAbs),initially identified in camelids or sharks and commonly referred to as nanobodies or VNARs,have emerged as a promising alternative to conventional therapeutic antibodies.These sdAbs have many superior physicochemical and pharmacological properties,including small size,good solubility and thermostability,easier accessible epitopes,and strong tissue penetration.However,the inherent challenges associated with the animal origin of sdAbs limit their clinical use.In recent years,various innovative humanization technologies,including complementarity-determining region(CDR)grafting or complete engineering of fully human sdAbs,have been developed to mitigate potential immunogenicity issues and enhance their compatibility.This review provides a comprehensive exploration of sdAbs,emphasizing their distinctive features and the progress in humanization methodologies.In addition,we provide an overview of the recent progress in developing drugs and therapeutic strategies based on sdAbs and their potential in solid tumor treatment,such as sdAbedrug conjugates,multispecific sdAbs,sdAb-based delivery systems,and sdAb-based cell therapy.

Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin

Type IV collagenase plays a pivotal role in invasion, metastasis and angiogenesis of tumor. Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size com-pared with antibody molecules produced by conventional methods. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic. In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type IV collagenase was prepared using a novel two-step method. First a VL-LDP fusion protein was constructed by DNA recombination. Secondly VL-LDP-AE was obtained by molecular reconstitution. In MTT assay, VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC50 values of 8.55×10-12 and 1.70×10-11 mol/L, respectively. VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane (CAM) assay and tube formation assay. In in vivo experiments, VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice. Drugs were given intravenously on day 3 and 10 after tumor transplantation. Compared in terms of maximal tolerated doses, VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%, LDM at 0.05 mg/kg by 69.9%, and mitomycin at 1 mg/kg by 35%. Having a molecular weight of 25.2 kDa, VL-LDP-AE was much smaller than other reported antibody-based drugs. The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy. And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.

Generation of HIY-resistant cells with a single-domain antibody:implications for HIV-1 gene therapy

The cure or functional cure of the"Berlin patient"and"London patient"indicates that infusion of HIV-resistant cells could be a viable treatment strategy.Very recently,we genetically linked a short-peptide fusion inhibitor with a glycosylphosphatidylinositol(GPI)attachment signal,rendering modified cells fully resistant to HIV infection.In this study,GPI-anchored m36.4,a single-domain antibody(nanobody)targeting the coreceptor-binding site of gp120,was constructed with a lentiviral vector.We verified that m36.4 was efficiently expressed on the plasma membrane of transduced TZM-bl cells and targeted lipid raft sites without affecting the expression of HIV receptors(CD4,CCR5,and CXCR4).Significantly,TZM-bl cells expressing GPI-m36.4 were highly resistant to infection with divergent HIV-1 subtypes and potently blocked HIV-1 envelope-mediated cell-cell fusion and cell-cell viral transmission.Furthermore,we showed that GPI-m36.4-modified human CEMss-CCR5 cells were nonpermissive to both CCR5-and CXCR4-tropic HIV-1 isolates and displayed a strong survival advantage over unmodified cells.It was found that GPI-m36.4 could also Impair HIV-1 Env processing and viral infectivity in transduced cells,underlying a multifaceted mechanism of antiviral action.In conclusion,our studies characterize m36.4 as a powerful nanobody that can generate HIV-resistant cells,offering a novel gene therapy approach that can be used alone or in combination.

Single-domain antibodies for radio nuclear imaging and therapy of esophageal squamous cell carcinoma:a narrative review

Single-domain antibodies have the characteristics of small molecular weight,strong tissue penetration,and high affinity,and are widely used to construct molecular probes for disease diagnosis and treatment.This article reviews molecular imaging studies including positron emission tomography(PET),single-photon emission computed tomography/computed tomography(CT),PET/CT,and fluorescent imaging of molecular probes composed of single-domain antibodies against eight esophageal squamous cell carcinoma biological targets.These 8 targets are highly expressed on the membrane of esophageal squamous cell carcinoma cells and include epidermal growth factor receptor,human epidermal growth factor receptor 2,human epidermal growth factor receptor 3,hepatocyte growth factor receptor,vascular endothelial growth factor receptor 2,chemokine receptor 4,chemokine receptor 7,and carcinoembryonic antigen.The current problems and solutions are also discussed to provide a reference for future design of molecular imaging probes targeting esophageal squamous cell carcinoma.

Co_(1-x)Zn_xFe_2O_4 nanofiber: Synthesized by electrospinning and its magnetic properties

Well-aligned and uniform Coo.sZno.2Fe204 nanofibers （NFs） are prepared by electrospinning via sol-gel and sub- sequent heat treatment. Each of the as-spun NFs has a diameter of about 300 nm and a smooth surface morphology. The scanning electron microscope （SEM） image shows that the diameter decreases to 70 nm after the Coo.sZno.2Fe204 NF has been annealed at 650℃ for 3 h. The structure and chemical of Co0.8Zn0.2Fe204 NF are investigated by X- ray diffraction （XRD） and energy dispersive X-ray spectroscopy （EDS）, respectively. Single phase cubic spinel structure, Coo.sZno.2Fe204 NF, is successfully obtained after having been calcined at 550 ~C in air for 3 h, and a reduced lattice constant of the Coo.8Zno.2Fe204 NF provides the evidence of effective Zn2＋ substitution. The magnetic measurements show that the substitution of Zn2＋ for Co2＋ , i.e., the introduction of non-magnetic Zn2＋ ions into A sites, can increase the saturation magnetization （Ms） and reduce the coercivity （He）. The obtained Hc results of different samples reveal that the critical single-domain size of the Co0.8Zn0.2Fe204 NF is approximately 44 nm. By doping Zn2＋ with different concentra- tions, the morphologies of Co1-xZnxFe2O4 （0 〈 x 〈 0.5） NFs do not show obvious changes. For magnetic properties, the Ms increases and Hc decreases monotonically, respectively.

Preparation and activity analysis of the divalent and tetravalent humanized V_H single domain antibody against human lung cancer

In order to improve the functional affinity of the humanized VH single domain antibody against human lung cancer, the genes coding the homogenous dimers dihu3D3Vn and tetramers tehu3D3VH were constructed by fusing the SV5-Cys short peptide and p53 tetramefization structural domain gene to hu3D3VH gene via recombinant PCR technique, respectively. Then, the dihu3D3VH and tehu3D3VH genes were cloned to the prokaryotic expression vector pET-22b（ ＋ ） and expressed in E. coli BL21 （DE3）. The proteins expressed were purified through Ni^2＋ -affinity chromatographic column. Meanwhile, the hu3D3VH, dihu3D3VH and tehu3D3VH proteins were labeled with FTTC, and their reactivity with antigen and specificity were analyzed by immunofluorescence assay. As to their functional affinities, it was analyzed and compared by flow cytometry. The results indicated that these two genes were expressed as monomers and mainly as inclusion bodies. After purification and renaturation, there were about 50% of dimers and 70% of tetramer remaining in the protein solution. In addition, the dihu3D3VH and tehu3D3VH proteins still remained the reactivity with antigen and specificity of hu3D3VH protein, and their functional affinities were increased about 60% or 100% respectively, compared with those of hu3D3VH protein. It is evident that the functional affinity of hu3D3VH protein can be greatly improved by increasing its binding valency.

Regulating photocatalytic overall water splitting of ferroelectric heterostructures by size effect

In the past decade,ferroelectric materials have been intensively explored as promising photocatalysts.An intriguing ability of ferroelectrics is to directly sperate the photogenerated electrons and holes,which is believed to arise from a spontaneous polarization.Understanding how polarization affects the photocatalytic performance is vital to design high-efficiency photocatalysts.In this work,we report a size effect of ferroelectric polarization on regulating the photocatalytic overall water splitting of SrTiO_(3)/PbTiO_(3)nanoplate heterostructures for the first time.This was realized hydrothermally by controlling the thickness and thus spontaneous polarization strength of single-crystal and single-domain PbTiO_(3)nanoplates,which served as the substrate for selective heteroepitaxial growth of SrTiO_(3).An enhancement of 22 times in the photocatalytic overall water splitting performance of the heterostructures has been achieved when the average thickness of the nanoplate increases from 30 to 107 nm.A combined experimental investigation revealed that the incompletely compensated depolarization filed is the dominated driving force for the photogenerated carrier separation within heterostructures,and its increase with the thickness of the nanoplates accounts for the enhancement of photocatalytic activity.Moreover,the concentration of oxygen vacancies for negative polarization compensation has been found to grow as the thickness of the nanoplates increases,which promotes oxygen evolution reaction and reduces the stoichiometric ratio of H_(2)/O_(2).These findings may provide the opportunity to design and develop high-efficiency ferroelectric photocatalysts.

A facile synthesis of magnetite single-crystal particles by employing GO sheets as template for promising application in magnetic fluid

It was reported a facile strategy to fabricate magnetite(Fe3O4)single-crystal particles with critical single-domain size by employing graphene oxide(GO)sheets as template.In this method,the small-sized Fe2 O3 nanoparticles were first synthesized,and then low-temperature annealing under H2 would convert them into large-sized Fe3 O4 single-crystal particles.The synthetic particles with an average size of 100 nm exhibit high saturation magnetization(Ms)of 0.085 A·m^2·g^-1,which is very close to theoretical value,being among the highest values in ever reported for Fe3O4 made from chemical methods.On this basis,the small-sized Fe3 O4 particles(average size of 30 nm)were also fabricated by coating with Na2 CO3 shell.

CALCULATION OF MAGNETIC DOMAIN STRUCTURE PARAMETERS IN Nd_2Fe_(14)B

I. INTRODUCTIONThe outstanding magnetic properties of Nd-Fe-B permanent alloy with very high coercive force and maximum magnetic energy product have attracted scientists’ attention greatly and extensive investigation has been made on its structure and mag-

Effect of Y_2Ba_4CuNbO_y on the Properties of Single Domain YBCO Superconductor by TSIG Process

The top-seeded infiltration and growth process (TSIG) is an important method to fabricate YBCO bulk superconductors. In this paper, single domain YBCO superconductors with different Gd2Ba4CuWOx (GdW2411) additions have been fabricated by TSIG process, the amount of GdW2411 added to Y2BaCuO5 (Y211) is in the range from 0wt% to 8 wt%, but there is no other additions to the liquid source. The growth morphology, microstructure and levitation force of the YBCO bulks have been investigated, it is found that the levitation force is increasing from 14 to 25 N with the increasing of GdW2411 addition from 0 to 4 wt%, and decreasing from 25 to 7 N with the increasing of GdW2411 addition from 4 to 8 wt%, the largest levitation force is obtained when the GdW2411 content is about 4 wt%. This is helpful for the fabrication of high quality YBCO bulk superconductors.