Evaluation of multi-specificity of antibody G2 using its single-chain Fv and its covalently linked antigen peptides.

The antibody G2 specifically binds to four peptides with different amino acid sequences: Pep18mer, Pep8, Pep395, and PepH4P6. To elucidate the multi-specificity of G2, we generated a G2 single-chain Fv (scFv) antibody and analyzed its binding thermodynamics and kinetics to antigen peptides. Our results clearly showed that the recognition of PepH4P6 was similar to that of Pep18mer, to which G2 could obtain binding ability through the deletion of Pro95 at light chain on the affinity maturation process. The covalent linking of peptides could increase the thermal stability of G2 scFv due to intramolecular antigen binding. In the effects of respective peptides, the increased thermal stability of G2 scFv linked to Pep8 was significant, possibly due to the rapid dissociation. Binding experiments of G2 scFv linked to peptides to other peptides showed decreased association rates relative to those of antigen-free G2 scFv while the dissociation rates were almost unchanged.

Strategies for selection and identification of rabbit single-chain Fv antibodies as ligand in affinity chromatography.

We developed affinity chromatographic resins that immobilized rabbit single-chain Fv antibodies (scFvs). By biopanning using antigen-coupled multilamellar vesicles (Ag-MLVs), 152 types of original scFv clones that specifically bind to human IgG were isolated and identified. Apparent dissociation rate constants, appkoff, of six different candidates were less than 10-3 s-1 and their dissociation constants, KDs, were ranged from 5.56 × 10-10 to 4.04 × 10-8 M. Consequently, the clones, R1-27, R2-18, and R3-26 were further investigated for use in affinity purification of human IgG. Both the clones, R1-27 and R3-26 maintained more than 40% of antigen-binding activities on the surface of affinity resins. Especially, R3-26 had a relatively high alkaline resistance. The direct separation of human IgG from 10% FBS-D-MEM by use of the column with R1-27 achieved 97.2% purity, while the column with R3-26 showed almost 100% recovery. The affinity resins at the densities between 4.32 and 15.19 mg-scFv/cm3 exhibited maximum binding amount of human IgG, while the highest ligand utilization was achieved by use of the resin at approximately 9 mg-scFv/cm3. The resin exhibited 7.69 mg/cm3 of equilibrium binding capacity (EBC) in affinity chromatography. It was expected that the EBC of affinity resins was strongly dependent on the specific surface area as well as the pore volume of the base resin. Therefore, the strategies to develop affinity ligands will be beneficial for development of on-demand affinity columns with higher affinity/selectivity, chemical resistance, while optimization of pore size and pore volume for scFv-coupled resins will further improve the EBC.

Gold nanoparticles and tilt pairs to assess protein flexibility by cryo-electron microscopy.

A computational method was developed to recover the three-dimensional coordinates of gold nanoparticles specifically attached to a protein complex from tilt-pair images collected by electron microscopy. The program was tested on a simulated dataset and applied to a real dataset comprising tilt-pair images recorded by cryo electron microscopy of RNA polymerase II in a complex with four gold-labeled single-chain antibody fragments. The positions of the gold nanoparticles were determined, and comparison of the coordinates among the tetrameric particles revealed the range of motion within the protein complexes.

Screen printed electrode-based biosensor functionalized with magnetic cobalt/single-chain antibody fragments for cocaine biosensing in different matrices.

On-site detection of substance abuse is an important approach in the preventive and intervention protocols implementations. It is known that the traditional methods are heavy, time-consuming, and need a high level of logistical requirements. As such, biosensors represent great potential to simplify and improve substance abuse detection. In this study, we have designed a functionalized screen-printed electrode (SPE) electrochemical biosensor with cobalt oxide nanoparticles and single-chain antibody fragments (scFvs) for cocaine detection. Different electrochemical techniques such as differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectrometry were used to examine the functionality of the designed biosensor. Furthermore, SEM observations were performed to observe the surface changes after functionalization. The results showed that the linearity ranged between 5.0 and 250 ng/mL and a detection limit of 3.6 ng/mL (n = 6). These results were compared to results obtained from Q-TOF/MS where four different matrices (serum, sweat, urine, and saliva) were spiked with 100 ng/mL cocaine and were analyzed by both methods (Biosensor and Q-TOF/MS). Results showed a higher performance of the biosensor compared to traditional methods. In addition, the selectivity of the biosensor was shown in the presence of different interferents where the designed platform showed a specific response to only cocaine. In conclusion, the designed biosensor proposes great potential for portable and on-site substance abuse detection in addition to boasting the capability of reuse of the SPE and thus, reducing the costs related to such applications.

Dendritic Cell Targeting Using a DNA Vaccine Induces Specific Antibodies and CD4+ T Cells to the Dengue Virus Envelope Protein Domain III.

Dengue fever has become a global threat, causing millions of infections every year. An effective vaccine against all four serotypes of dengue virus (DENV) has not been developed yet. Among the different vaccination strategies available today, DNA vaccines are safe and practical, but currently induce relatively weak immune responses in humans. In order to improve immunogenicity, antigens may be targeted to dendritic cells (DCs), the main antigen presenting cells and orchestrators of the adaptive immune response, inducing T and B cell activation. It was previously shown that a DNA vaccine encoding a fusion protein comprised of an antigen and a single-chain Fv antibody (scFv) specific for the DC endocytic receptor DEC205 induced strong immune responses to the targeted antigen. In this work, we evaluate this strategy to improve the immunogenicity of dengue virus (DENV) proteins. Plasmids encoding the scFv αDEC205, or an isotype control (scFv ISO), fused to the DENV2 envelope protein domain III (EDIII) were generated, and EDIII specific immune responses were evaluated in immunized mice. BALB/c mice were intramuscularly (i.m.) immunized three times with plasmid DNAs encoding either scDEC-EDIII or scISO-EDIII followed by electroporation. Analyses of the antibody responses indicated that EDIII fusion with scFv targeting the DEC205 receptor significantly enhanced serum anti-EDIII IgG titers that inhibited DENV2 infection. Similarly, mice immunized with the scDEC-EDIII plasmid developed a robust CD4+ T cell response to the targeted antigen, allowing the identification of two linear epitopes recognized by the BALB/c haplotype. Taken together, these results indicate that targeting DENV2 EDIII protein to DCs using a DNA vaccine encoding the scFv αDEC205 improves both antibody and CD4+ T cell responses. This strategy opens perspectives for the use of DNA vaccines that encode antigens targeted to DCs as a strategy to increase immunogenicity.

Discovery of internalizing antibodies to basal breast cancer cells.

We present a strategy to discover recombinant monoclonal antibodies (mAbs) to specific cancers and demonstrate this approach using basal subtype breast cancers. A phage antibody library was depleted of antibodies to common cell surface molecules by incubation with luminal breast cancer cell lines, and then selected on a single basal-like breast cancer cell line (MDA-MB-231) for binding associated receptor-mediated endocytosis. Additional profiling against two luminal and four basal-like cell lines revealed 61 unique basal-specific mAbs from a pool of 1440 phage antibodies. The unique mAbs were further screened on nine basal and seven luminal cell lines to identify those with the greatest affinity, specificity, and internalizing capability for basal-like breast cancer cells. Among the internalizing basal-specific mAbs were those recognizing four transmembrane receptors (EphA2, CD44, CD73 and EGFR), identified by immunoprecipitation-mass spectrometry and yeast-displayed antigen screening. Basal-like breast cancer expression of these four receptors was confirmed using a bioinformatic approach, and expression microarray data on 683 intrinsically subtyped primary breast tumors. This overall approach, which sequentially employs phage display antibody library selection, antigen identification and bioinformatic confirmation of antigen expression by cancer subtypes, offers efficient production of high-affinity mAbs with diagnostic and therapeutic utility against specific cancer subtypes.

Bovine single chain Fv antibody inhibits bovine herpesvirus-1 infectivity by targeting viral glycoprotein D.

Glycoprotein D (gD) of bovine herpesvirus-1 (BoHV-1) is essential for attachment and penetration of cells during infection and is a major target for neutralizing antibodies during an adaptive immune response. Currently there are no recombinant antibodies capable of binding gD epitopes for use in treating BoHV-1 infection. In this study, a bovine scFv gene derived from a hybridoma secreting monoclonal antibodies (McAbs) against the amino acid motif MEESKGYEPP of gD was expressed in E. coli. Molecular modeling, western blot and ELISA analysis showed that this scFv had a high affinity for BoHV-1 gD, with a Kd of 161.2 ± 37.58 nM and for whole BoHV-1 virus, with a Kd of 67.44 ± 16.99 nM. In addition, this scFv displayed a high affinity for BoHV-1 antigen in an ELISA and competed with BoHV-1 anti-serum in a competitive ELISA. Immunofluorescence assay (IFA) and laser confocal microscopy showed that this scFv could efficiently bind to and be internalized by BoHV-1 infected Madin-Darby bovine kidney (MDBK) cells. Importantly, this scFv was shown to inhibit BoHV-1 infectivity and to reduce the number of viral plaques by blocking viral attachment to MDBK cells. Our study suggests that this bovine single-chain antibody could be developed for use as a diagnostic and therapeutic agent against BoHV-1 infection in cattle.

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments.

Expression, purification and biological effect of a novel single chain Fv antibody and protamine fusion protein for the targeted delivery of siRNAs to FGFR3 positive cancer cells

Background: Gain-of-function of fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies have focused on the potential usage of therapeutic single-chain Fv (ScFv) antibodies against FGFR3. RNA interference (RNAi) has been considered as a promising therapeutic method against cancer. A tool which can deliver small interference RNAs (siRNAs) into FGFR3 positive cancer cells is very promising for anti-tumor therapy. Results: In this study, a novel fusion protein R3P, which consists of FGFR3-ScFv and protamine, was generated in Escherichia coli by inclusion body expression strategy and Ni-NTA chromatography. Its yield reached 10 mg per liter of bacterial culture and its purity was shown to be higher than 95%. 1 µg of R3P could efficiently bind to about 2.5 pmol siRNAs and deliver siRNAs into FGFR3 positive RT112 and K562 cells. Annexin V staining results showed that R3P can deliver the amplified breast cancer 1 (AIB1) siRNAs to induce RT112 cell apoptosis. Conclusion: These results indicated that R3P was a promising carrier tool to deliver siRNAs into FGFR3 positive cancer cells and to exert anti-tumor effect.

Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy.

Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%-347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.

Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli

Background Overexpression or mutated activation of Fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies focus on the potential usage of therapeutic antibodies against FGFR3. Results In this study, a novel single-chain Fv (ScFv) against FGFR3 was prepared and characterized. To achieve the soluble expression, ScFv was fused with Sumo (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and cloned into pET-20b. The recombinant bacteria were induced by 0.5 mM Isopropyl-ß-d-thiogalactopyranoside (IPTG) for 16 h at 20°C, and the supernatant liquid of Sumo-ScFv was harvested and purified by Ni-NTA chromatography. After being cleaved by the Sumo protease, the recombinant ScFv was released from the fusion protein, and further purified by Ni-NTA chromatography. The purity of ScFv was shown to be higher than 95% and their yield reached 4 mg per liter of bacterial culture. In vitro data showed that ScFv can significantly attenuate FGF9-induced phosphorylation of FGFR3. Conclusion We provide a novel method to produce soluble expression and bioactive functions of ScFv in Escherichia coli.

Using the local immune response from the natural buffalo host to generate an antibody fragment library that binds the early larval stages of Schistosoma japonicum.

Antibodies isolated from the local draining inguinal lymph node of field exposed-water buffaloes following challenge with Schistosoma japonicum cercariae showed high reactivity towards S. japonicum antigen preparations and bound specifically to formaldehyde-fixed S. japonicum schistosomules. Using this specific local immune response we produced a series of single-chain antibody Fv domain libraries from the same lymph nodes. Removal of phage that cross reacted with epitopes on adult parasites yielded a single-chain antibody Fv domain-phage library that specifically bound to whole formaldehyde-fixed and live S. japonicum schistosomules. DNA sequencing indicated clear enrichment of the single-chain antibody Fv domain library for buffalo B-cell complementarity determining regions post-selection for schistosomule binding. This study also revealed that long heavy chain complementarity determining regions appear to be an important factor when selecting for antibody binding fragments against schistosomule proteins. The selected single-chain antibody Fv domain-phage were used to probe a schistosome-specific protein microarray, which resulted in the recognition of many proteins expressed across all schistosome life-cycle stages. Following absorption to adult worms, the single-chain antibody Fv domain-phage library showed significantly reduced binding to most proteins, whilst two proteins (NCBI GenBank accession numbers AY915878 and AY815196) showed increased binding. We have thus developed a unique set of host derived single-chain antibody Fv domains comprising buffalo B-cell variable regions that specifically bind to early S. japonicum life-stages.

Structure of a single-chain Fv bound to the 17 N-terminal residues of huntingtin provides insights into pathogenic amyloid formation and suppression.

Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3-11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12-17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a ß-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv-peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT.

Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli.

The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10(-8) mol·L(-1), its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10(-7) mol·L(-1). The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L(-1).

Anti-hepatoma human single-chain Fv antibody and adriamycin conjugates with potent antitumor activity.

To construct an improved biological missile, an immunoconjugate ADM-Dex-ScFv-SA3 was synthesized, which was composed of a hepatocellular carcinoma-specific, single-chain Fv antibody (ScFv-SA3) and a highly potent cytotoxic drug, adriamycin (ADM), as the warhead. Oxidized Dextran T10 (Dex-T10) was used as a linker to connect these two moieties. The 40 KD soluble anti-hepatoma human Trx-ScFv-SA3 protein was expressed in E. coli BL21 (DE3), using a prokaryotic expression vector, pET21a (+)-Trx-ScFv-SA3-His. It was purified using a His-Tag Ni-Agarose column and identified by western blot. The activity of Trx-ScFv-SA3 was verified by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry to confirm that it specifically binds to the hepatocellular carcinoma cell line HepG2. To prepare ADM-Dex-ScFv-SA3, ADM was conjugated to the antibody at a molar ratio of 14.21:1. The antitumor effect of the conjugate was tested by MTT assay, plate colony formation assay and xenografts in a nude mice experimental model. In vitro experiments revealed that ADM-Dex-ScFv-SA3 could bind to tumor cells selectively and inhibit the proliferation and the colony formation ability of HepG2 cells. In vivo experiments showed that ADM-Dex-ScFv-SA3 suppressed the tumor growth and prolonged the median survival time in tumor-bearing mice. Tumor histology slides indicated a significantly slower tumor tissue proliferation in the ADM-Dex-ScFv-SA3 group. These data indicate that the targeted drug, ADM-Dex-ScFv-SA3, may be a highly potent and selective therapy for the treatment of hepatoma.

Preparation and Identification of Anti-lactoferrin Single Chain Fv Antibody / 医学研究杂志

Objective To prepare a specific anti-lactoferrin single chain variable fragment(ScFv).Methods Anti-lactoferrin clones were screened from a 'naive' phage antibody library against the immobilized lactoferrin antigen,then the clones were transformed to the E.coli HB2151 to give soluble expression of antibody fragments.The culture supernatant containing ScFv was purified by immobilized metal affinity chromatography,and then determined with SDS-PAGE and ELISA.Results The results demonstrated that ScFvs were specific;they did not react with transferrin,lysozyme and bovine serum albumin in ELISA.The SDS-PAGE showed that the ScFvs had high purity through affinity chromatography and the molecular weight of them was about 32 kD.Conclusions The successful generation of the ScFvs against lactoferrin provides a basis for further study and clinical applications for dry eye and other ocular diseases.

Nanocomoposite probes composed of fluorescent magnetic nanoparticles and PSA ScFv antibody for targeted imaging and therapy of prostate cancer / 中国肿瘤生物治疗杂志

Objective:To investigate the feasibility of targeted imaging and therapy of prostate cancer using nanocomposite probes composed of fluorescent magnetic nanoparticles(FMCNPs) and single chain Fv(ScFv) antibody specific for gama-seminoprotein.Methods:The nanocomposite probes(FMCNPs-ScFv) were prepared by conjugating fluorescent magnetic nanoparticles with singlegama-chain Fv antibody specific gama-seminoprotein,and were characterized by high resolution transmission electron microscopy,fluorescent spectrum and magnetic spectrum.Nanocomposite probes were incubated with prostate cancer LNCaP cells,and the targeting results of nanocomposite probes were observed by fluorescent microscopy.The cytotoxicity effect of the nanocomposite probes was measured by MTT.Nude mice models of prostate cancer were established and identified by immunohistochemistry method.The nanocomposite probes were injected into nude mice via tail vein.The distribution of nanocomposite probes in the nude mice was observed by Micro-animal imaging system,targeted imaging of the prostate cancer was observed by MR instrument.The nude mice with prostate cancer were irradiated with 100 W magnetic field for 30 min,and the changes of tumor sizes were observed.Results:The FMCNPs-ScFv nanocomposite probes were successfully prepared.Nanocomposite probes entered into the cytoplasm of cancer cells and exhibited low cytotoxicity effect.Nude mice model with prostate cancer were successfully fabricated;the nanocomposite probes distributed quickly in the main organs of mice,and gradually concentrated on the tumor tissues within 24 h.MR images showed that the tumor images were gradually enhanced from 6 h to 24 h after injection of the nanocomposite probe.Four days after magnetic irradiation,the tumors in the nude mice grew slower compared with the control nude mice(P

Screening and identification of anti-HRPII single chain Fv antibodies of Plasmodium falciparum / 细胞与分子免疫学杂志

To develop simple, rapid, and efficacious diagnostic methods for malaria is one of the remaining key tasks for malaria control. Previously, we have created a phage-displayed antibody library against Plasmodium falciparum. Six clones of antibody with good reactivity to HRP-II in ELISA were isolated from the library after 3 rounds of enrichment. Soluble ScFvs were produced and the characteristics were determined. The results of Western blot showed that they could bind to HRP-II specifically and had a relative molecular mass(Mr) about 31 000. The work provided a solid fund for diagnostic kit development for malaria.

Preparation and immunohistochemical study of human single chain Fv antibody against hepatitis C virus core antigen / 解放军医学杂志

Objective To detect hepatitis C virus core antigen in 7721 cells transfected with HCV cDNA by immunohistochemistry method with human single chain Fv antibody(scFv). Methods The recombinant phages were panned by core antigen which was coated in a microtiter plate. After three rounds of biopanning, 48 clones were identified specific to core antigen. The affinity and specificity of scFv were evaluated by ELISA and immunohistochemistry. Results ScFv-core DNA digestion and sequence data showed that the scFv gene was composed of 774bp. Conclusion Human single chain Fv antibody against HCV core antigen has a specific combining capacity with hepatitis C virus core antigen.

EXPRESSION OF SOLUBLE HUMAN ScFv AGAINST CORE PROTEIN OF HEPATITIS C VIRUS IN E COLI / 解放军医学杂志

To construct expressive vector for human ScFv against core protein of hepatitis C virus (HCV core ScFv),and to express soluble HCV core ScFv in E.coli JM109. Using phage display technique, the recombinant phages were panned by recombinant core antigen which was coated in a microtiter plate, and after five rounds of biopanning, 86 clones were identified specific to core antigen. 750 bp fragment could be released from the plasmid of positive phage clones, and the sequence analysis indicated that we have obrained the ScFv DNA fragment. Then DNA fragment was inserted into the expressive vector pCANTAB5E, and E. coli host JM109 was transformed and induced by IPTG. The specificity of ScFv in the culture medium was evaluated by enzyme linked immunosorbent assay (ELISA).The molecular weight of expressed HCV core ScFv protein is 28 000 dalton as determined with the aid of SDS polyacrymide gel electrophoresis (PAGE). The expressed HCV core ScFv protein will be useful in the immunohistochemical study of liver tissue from patients with hepatitis C and gene therapy against HCV infection.