scholarly journals Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2311
Author(s):  
Kohei Yumoto ◽  
Tomoaki Arisaka ◽  
Kazuma Okada ◽  
Kyosuke Aoki ◽  
Toyoyuki Ose ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Expression System ◽  
Case Fatality ◽  
Baculovirus Expression System ◽  
Amino Acid Sequences ◽  
Single Chain ◽  
Neutralizing Activity ◽  
Binding Characteristics ◽  
Single Chain Fv

Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previously established mouse monoclonal antibodies, 15–13 and 12–22, which showed neutralizing activity against the RABV, targeting the sequential and conformational epitopes on the RABVG, respectively. However, the molecular basis for the neutralizing activity of these antibodies is not yet fully understood. In this study, we evaluated the binding characteristics of the Fab fragments of the 15–13 and 12–22 antibodies. The recombinant RABVG protein, in prefusion form for the binding analysis, was prepared by the silkworm–baculovirus expression system. Biolayer interferometry (BLI) analysis indicated that the 15–13 Fab interacts with the RABVG, with a KD value at the nM level, and that the 12–22 Fab has a weaker binding affinity (KD ~ M) with the RABVG compared to the 15–13 Fab. Furthermore, we determined the amino acid sequences of both the antibodies and the designed single-chain Fv fragments (scFvs) of the 15–13 and 12–22 antibodies as another potential biopharmaceutical for targeting rabies. The 15–13 and 12–22 scFvs were successfully prepared by the refolding method and were shown to interact with the RABVG at the nM level and the µM level of the KD, respectively. These binding characteristics were similar to that of each Fab. On the other hand, differential scanning fluorometry (DSF) revealed that the thermal stability of these scFvs decreases compared to their Fabs. While the improvement of the stability of scFvs will still be required, these results provide insights into the neutralizing activity and the potential therapeutic use of antibody fragments for RABV infection.

scholarly journals Generation and Characterization of an scFv Directed against Site II of Rabies Glycoprotein

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Shukra M. Aavula ◽  
Sridevi V. Nimmagadda ◽  
Neelakantam Biradhar ◽  
Samuel Sula ◽  
Dev Chandran ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Specific Binding ◽  
Recombinant Antibody ◽  
Antigenic Site ◽  
Antibody Fragment ◽  
Single Chain ◽  
Phage Display Technology ◽  
Single Chain Fv ◽  
Rabies Glycoprotein

Recombinant antibody phage display technology is a vital tool that facilitates identification of specific binding molecules to a target enabling the rapid generation and selection of high affinity, fully human, or mouse antibody product candidates essentially directed towards disease target appropriate for antibody therapy. In this study, a recombinant single-chain Fv antibody fragment (scFv) A11 was isolated from immune spleen cells obtained from mice immunized with inactivated rabies virus (Pasteur strain) using standard methodology and was characterized for its specificity towards the rabies virus glycoprotein. Epitope mapping using peptide libraries and truncated glycoprotein polypeptides suggested that A11 bound to the antigenic site II of rabies glycoprotein against which a majority of rabies virus neutralizing antibodies are directed. The use of the above technology could, therefore, allow development of scFvs with different specificities against the rabies glycoprotein as an alternative to the more cumbersome protocols used for the development of monoclonal antibodies.

scholarly journals Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli

2022 ◽  
Vol 44 (1) ◽  
pp. 301-308
Author(s):  
Sun-Hee Kim ◽  
Hee-Jin Jeong
Keyword(s):  
Fusion Protein ◽  
Expression System ◽  
Antibody Fragment ◽  
Therapeutic Index ◽  
High Yield ◽  
Soluble Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Fv ◽  
Programmed Death

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression

2020 ◽  
Vol 168 (3) ◽  
pp. 257-263 ◽  
Author(s):  
Chenjiang Liu ◽  
Yoshihiro Kobashigawa ◽  
Soichiro Yamauchi ◽  
Natsuki Fukuda ◽  
Takashi Sato ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Low Cost ◽  
Recombinant Antibody ◽  
Expression System ◽  
Single Chain ◽  
Application Study ◽  
Variable Regions ◽  
Protein Ligation ◽  
Split Intein ◽  
Single Chain Fv

Abstract Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH–VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

scholarly journals Isolation of Human Monoclonal Antibodies That Neutralize Human Rotavirus

2004 ◽  
Vol 78 (7) ◽  
pp. 3325-3332 ◽  
Author(s):  
Kyoko Higo-Moriguchi ◽  
Yasushi Akahori ◽  
Yoshitaka Iba ◽  
Yoshikazu Kurosawa ◽  
Koki Taniguchi
Keyword(s):  
Neutralizing Antibodies ◽  
Cross Reactivity ◽  
Amino Acid Sequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Antibody ◽  
Display System ◽  
Neutralizing Activity ◽  
Human Antibodies ◽  
Human Rotavirus ◽  
Human Specific

ABSTRACT A human antibody library constructed by utilizing a phage display system was used for the isolation of human antibodies with neutralizing activity specific for human rotavirus. In the library, the Fab form of an antibody fused to truncated cp3 is expressed on the phage surface. Purified virions of strain KU (G1 serotype and P[8] genotype) were used as antigen. Twelve different clones were isolated. Based on their amino acid sequences, they were classified into three groups. Three representative clones—1-2H, 2-3E, and 2-11G—were characterized. Enzyme-linked immunosorbent assay with virus-like particles (VLP-VP2/6 and VLP-VP2/6/7) and recombinant VP4 protein produced from baculovirus recombinants indicated that 1-2H and 2-3E bind to VP4 and that 2-11G binds to VP7. The neutralization epitope recognized by each of the three human antibodies might be human specific, since all of the antigenic mutants resistant to mouse monoclonal neutralizing antibodies previously prepared were neutralized by the human antibodies obtained here. After conversion from the Fab form of an antibody into immunoglobulin G1, the neutralizing activities of these three clones toward various human rotavirus strains were examined. The 1-2H antibody exhibited neutralizing activity toward human rotaviruses with either the P[4] or P[8] genotype. Similarly, the 2-3E antibody showed cross-reactivity against HRVs with the P[6], as well as the P[8] genotype. In contrast, the 2-11G antibody neutralized only human rotaviruses with the G1 serotype. The concentration of antibodies required for 50% neutralization ranged from 0.8 to 20 μg/ml.

scholarly journals Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

2015 ◽  
Vol 112 (32) ◽  
pp. 9860-9865 ◽  
Author(s):  
Changming Yu ◽  
Gennett M. Pike ◽  
Tommy A. Rinkoski ◽  
Cristina Correia ◽  
Scott H. Kaufmann ◽  
...  
Keyword(s):  
Large Scale ◽  
Expression System ◽  
Protein Translation ◽  
Avian Leukosis Virus ◽  
Eukaryotic Expression ◽  
Eukaryotic Cells ◽  
Single Chain ◽  
Mammalian Expression ◽  
Single Chain Fv ◽  
Fv Antibody

Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.

scholarly journals Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3393-3399 ◽  
Author(s):  
Gregory Kanter ◽  
Junhao Yang ◽  
Alexei Voloshin ◽  
Shoshana Levy ◽  
James R. Swartz ◽  
...  
Keyword(s):  
B Cell ◽  
Expression System ◽  
Specific Antigen ◽  
Therapeutic Vaccine ◽  
Antibody Fragment ◽  
B Cell Lymphoma ◽  
Keyhole Limpet Hemocyanin ◽  
Single Chain ◽  
Single Chain Fv ◽  
Fv Antibody

Abstract The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli—based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

scholarly journals Neutralization Breadth and Potency of Single-Chain Variable Fragments Derived from Broadly Neutralizing Antibodies Targeting Multiple Epitopes on the HIV-1 Envelope

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Rebecca T. van Dorsten ◽  
Bronwen E. Lambson ◽  
Constantinos Kurt Wibmer ◽  
Marc S. Weinberg ◽  
Penny L. Moore ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Passive Immunization ◽  
Loss Of Function ◽  
Single Chain ◽  
Broadly Neutralizing Antibodies ◽  
Variable Regions ◽  
Neutralizing Activity ◽  
Single Chain Variable Fragments ◽  
Key Residues ◽  
Hiv 1

ABSTRACT Passive administration of HIV-directed broadly neutralizing antibodies (bNAbs) can prevent infection in animal models, and human efficacy trials are under way. Single-chain variable fragments (scFv), comprised of only the variable regions of antibody heavy and light chains, are smaller molecules that may offer advantages over full-length IgG. We designed and expressed scFv of HIV bNAbs prioritized for clinical testing that target the V2-apex (CAP256-VRC26.25), V3-glycan supersite (PGT121), CD4 binding site (3BNC117), and MPER (10E8v4). The use of either a 15- or 18-amino-acid glycine-serine linker between the heavy- and light-chain fragments provided adequate levels of scFv expression. When tested against a 45-multisubtype virus panel, all four scFv retained good neutralizing activity, although there was variable loss of function compared to the parental IgG antibodies. For CAP256-VRC26.25, there was a significant 138-fold loss of potency that was in part related to differential interaction with charged amino acids at positions 169 and 170 in the V2 epitope. Potency was reduced for the 3BNC117 (13-fold) and PGT121 (4-fold) scFv among viruses lacking the N276 and N332 glycans, respectively, and in viruses with a longer V1 loop for PGT121. This suggested that scFv interacted with their epitopes in subtly different ways, with variation at key residues affecting scFv neutralization more than the matched IgGs. Remarkably, the scFv of 10E8v4 maintained breadth of 100% with only a minor reduction in potency. Overall, scFv of clinically relevant bNAbs had significant neutralizing activity, indicating that they are suitable for passive immunization to prevent HIV-1 infection. IMPORTANCE Monoclonal antibodies have been isolated against conserved epitopes on the HIV trimer and are being investigated for passive immunization. Some of the challenges associated with full-sized antibody proteins may be overcome by using single-chain variable fragments (scFv). These smaller forms of antibodies can be produced more efficiently, may show fewer off-target effects with increased tissue penetration, and are more adaptable to vectored-mediated expression than IgG. Here, we demonstrate that scFv of four HIV-directed bNAbs (CAP256-VRC26.25, PGT121, 3BNC117, and 10E8v4) had significant neutralizing activity against diverse global strains of HIV. Loss of potency and/or breadth was shown to be due to increased dependence of the scFv on key residues within the epitope. These smaller antibody molecules with functional activity in the therapeutic range may be suitable for further development as passive immunity for HIV prevention.

Identification of Binding Epitope for Anti-Rabies Virus Glycoprotein Single-Chain Fv Fragment FV57

2011 ◽  
Vol 18 (11) ◽  
pp. 1099-1106 ◽  
Author(s):  
Tie-jun Gu ◽  
Wei Wei ◽  
Ye Duan ◽  
Chun-lai Jiang ◽  
Yan Chen ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Single Chain ◽  
Rabies Virus Glycoprotein ◽  
Virus Glycoprotein ◽  
Single Chain Fv ◽  
Binding Epitope ◽  
Single Chain Fv Fragment

scholarly journals Construction of a Functional Single-Chain Variable Fragment Antibody against Hemagglutinin from Porphyromonas gingivalis

1998 ◽  
Vol 66 (5) ◽  
pp. 2207-2212 ◽  
Author(s):  
Yasuko Shibata ◽  
Kimiyo Kurihara ◽  
Hisashi Takiguchi ◽  
Yoshimitsu Abiko
Keyword(s):  
Porphyromonas Gingivalis ◽  
Recombinant Antibody ◽  
Expression System ◽  
Passive Immunization ◽  
Amino Acid Sequences ◽  
Hemagglutinating Activity ◽  
Bacterial Invasion ◽  
Nucleotide Sequencing ◽  
Single Chain Variable Fragment ◽  
Single Chain

ABSTRACT Hemagglutinin is a major glycoprotein of Porphyromonas gingivalis vesicles and likely confers the ability to adsorb and penetrate into host tissue cells. To protect this bacterial invasion, murine monoclonal antibody (MAb) Pg-vc, which inhibited the hemagglutinating activity, was prepared by using P. gingivalis vesicles as an antigen. Western blot analysis revealed that when both MAb Pg-vc and anti-HA-Ag2 antibody raised against theP. gingivalis hemagglutinin adhesin (M. Deslauriers and C. Mouton, Infect. Immun. 60:2791–2799, 1992) were allowed to react with protein blots from P. gingivalis vesicles, a superimposable profile was observed. To obtain a recombinant antibody, cDNAs coding for the variable domains of the L and H chains of MAb Pg-vc were cloned by PCR, and a plasmid specifying a single-chain variable fragment (ScFv) was constructed. Following transformation of Escherichia coli cells, a recombinant ScFv protein was successfully expressed. The immunological properties of this protein were identical to those of the parental murine MAb, specifically recognizing the two proteins (43 and 49 kDa) originating from P. gingivalisvesicles. In addition, the ScFv antibody inhibited theP. gingivalis vesicle-associated hemagglutinating activity. The amino acid sequences deduced from nucleotide sequencing experiments confirmed that variable heavy-chain and variable light-chain regions belonged to VH1 and Vκ12/13 families, respectively. Since the expression system used in this study can readily provide large quantities of single-chain recombinant antibody, it may be a useful in developing a therapeutic agent for passive immunization in humans.

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