Cloning,expression, and identification of anti‐carbofuran single chain Fv gene

Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly₄Ser)₃. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC₅₀ value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

An anti-leukemic single chain Fv antibody selected from a synthetic human phage antibody library

The display of human antibody repertoire on the cell surface of the filamentous bacteriophage has offered a novel strategy for selecting antibodies to a diverse range of purified targets. However, the selection of antibodies with biological functions has not yet been fully investigated. To select phage antibodies with therapeutic potential, a synthetic human single chain Fv (scFv) phage antibody library was panned on whole premyelocytic leukemia cell line (HL60). Phages binding to common receptors and undesirable phages were subtracted by incubating the library with human glioma cells. High affinity binding phages to HL60 cells were enriched by fluorescence-activated cell sorting. After the 6th round of selection, 50% of the selected phage antibodies showed significant binding to HL60 cells, whereas none of the analyzed phage antibodies bound to human pre-B cells (Nalm-6). In addition to binding, one scFv antibody inhibited HL60 cell proliferation by 90% compared to irrelevant scFv antibodies. Taken together the data demonstrate that specific scFv antibodies with biological functions can be isolated by using whole cells as affinity matrix.     

Expression and Characterization of a Functional Single-Chain Variable Fragment (scFv) Protein Recognizing MCF7 Breast Cancer Cells in E. coli Cytoplasm

Single-chain variable fragment (scFv) is one of the most common antibody forms. This report describes the expression of the scFv gene as a soluble protein in Origami DE3 cytoplasm. The purified scFv recognized the epidermal growth factor receptor (EGFRvIII) on the surface of MCF-7 cells. The scFv protein was purified in soluble form at a concentration of 10 mg/l, and the scFv protein activity and specificity were characterized using several immunological assays. The purified scFv protein showed specific binding to MCF-7 cells, evidenced by a band of 68 kDa in Western blot analysis, and immunofluorescence clearly proved that the scFv antibody recognized the EGFRvIII antigen epitopes. Furthermore, 53 % of the MCF-7 cells were bound to scFv protein, as measured by flow cytometry analysis. This study demonstrated that the Origami DE3 expression system can produce single-chain antibodies in active form for later use in gene therapy and vaccine production.     

An efficient route to the production of an IgG-like bispecific antibody

Production of IgG-form bispecific antibody (BsAb-IgG) by co-expressing two antibodies in transfected cells is often inefficient owing to the unwanted pairing between the component heavy and light chains. We have developed an efficient method for the production of a novel IgG-like BsAb by using the natural dimerization mechanism between IgG heavy and light chains. Two single-chain Fv (scFv) of different specificity are fused to the constant domain of human kappa chain (C(L)) and the first constant domain of human heavy chain (C(H1)), to form two polypeptides, (scFv)(1)-C(L) and (scFv)(2)-C(H1)-C(H2)-C(H3), respectively. Co-expression of the two polypeptides in mammalian cells results in the formation of a covalently linked IgG-like hetero-tetramer, Bs(scFv)(4)-IgG, with dual specificity. Our approach yields a homogeneous bispecific IgG-like antibody product with each molecule containing four antigen binding sites, two for each of its target antigens. A Bs(scFv)(4)-IgG was prepared using two scFv antibodies each directed against a different epitope of a vascular endothelial growth factor receptor, the kinase insert domain-containing receptor (KDR). The Bs(scFv)(4)-IgG is capable of simultaneously binding to the two epitopes on the receptor. Further, the Bs(scFv)(4)-IgG also retains the antigen-binding efficacy and biological activity of its component antibodies.     

Production and characterization of a humanized single-chain antibody against human integrin alphav beta3 protein

Anti-angiogenesis therapy is an emerging strategy for cancer treatment. This therapy has many advantages over existing treatments, such as fewer side effects, fewer resistance problems, and a broader tumor type spectrum. Integrin αvβ3 is a heterodimeric transmembrane glycoprotein that has been demonstrated to play a key role in tumor angiogenesis and metastasis. We have used a phage antibody display to humanize a mouse monoclonal antibody (mAb E10) against human integrin αvβ3 with a predetermined CDR3 gene. Three human phage antibodies were developed. Analysis of the humanized phage antibodies by phage ELISA revealed that the antibodies retained high antigen-binding activity and detected the same epitope as the parent mAb E10. A humanized single chain Fv (scFv) antibody was expressed in Escherichia coli in a soluble form. Analysis of the purified scFv indicated that it has the same specificity and affinity as the original mAb. Cell viability assays and xenograft model results suggested that the humanized scFv possesses anti-tumor growth activity in vitro and in vivo. This successful production of a humanized scFv with the ability to inhibit αvβ3-mediated cancer cell growth may provide a novel candidate for integrin αvβ3-targeted therapy.     

Production and characterization of a CD25-specific scFv-Fc antibody secreted from Pichia pastoris

Antibodies against CD25 would be novel tools for the diagnosis and treatment of adult T cell leukemia lymphoma (ATLL) and many other immune disorders. In our previous work, we successfully produced the single-chain fragment of a variable antibody against CD25, the Dmab(scFv) antibody, using Pichia pastoris. Here, we describe a novel form of an antibody against CD25, the Dmab(scFv)-Fc antibody, also produced by P. pastoris. To construct the Dmab(scFv)-Fc antibody, the Dmab(scFv) antibody was genetically fused to the Fc fragment of a human IgG1 antibody. A fusion gene encoding Dmab(scFv)-Fc antibody was cloned into the pPIC9K plasmid and expressed at high levels, 60–70 mg/l, by P. pastoris under optimized conditions. The Dmab(scFv)-Fc antibody was similar to the Dmab(scFv) antibody in its binding specificity but different in its molecular form and Fc-mediated effector functions. The Dmab(scFv)-Fc antibody and the Dmab(scFv) antibody both bound to CD25-positive MJ cells but not to CD25-negative K562 cells. The Dmab(scFv)-Fc antibody existed as a dimer whereas the Dmab(scFv) antibody was a monomer because it lacks the Fc fragment. The Dmab(scFv)-Fc antibody enhanced the antibody-dependent cellular cytotoxicity of CD25-positive cancer cells, whereas the Dmab(scFv) antibody was inactive in the antibody-dependent cellular cytotoxicity assays. In addition, compared to the Dmab(scFv) antibody, the Dmab(scFv)-Fc antibody showed stronger immunosuppressive activity in the Con A-stimulated lymphocyte proliferation system and in the mixed lymphocyte reaction system. These results demonstrate that the Dmab(scFv)-Fc antibody produced in P. pastoris is functional, and therefore it might be developed as a novel diagnostic and therapeutic tool for ATLL and other immune disorders.     

Phage displayed antibodies to heat stable alkaline phosphatase: framework region as a determinant of specificity

Human antibodies against specific targets of tumor cells are the most desirable molecules for possible immunotherapy. They could be developed by using the combinatorial antibody library displayed on a phage. We selected four human antibody fragments (scFv) binding to the oncoplacental antigen Heat Stable Alkaline Phosphatase (HSAP, the placental isozyme of alkaline phosphatase) from a synthetic human antibody library. Characterization of these scFvs showed they bound HSAP with moderate affinity but did not have isozyme specificity, as determined by binding to cell lines exhibiting differential expression of isozymes of alkaline phosphatase. The V(H) sequences of two of these scFvs were similar and although both bound to HSAP only one was cross-reactive with albumin. The sequences revealed a difference in the framework region (FR1) of these antibodies, indicating a role for this region in the determination of specificity. This is also significant considering that the heavy chains generated the diversity of the synthetic library used in this study, and only a single light chain showing binding to BSA was used for the entire library.     

A rapid novel strategy for screening of antibody phage libraries for production,purification, and functional characterization of amber stop codons containing single-chain antibody fragments

Phage display antibody (PDA) libraries, allows the rapid isolation and characterization of high specificity monoclonal antibodies for therapeutic and diagnostic applications. However, selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias towards clones containing randomly generated amber stop codons, complicating the identification of high affinity binding antibodies. We screened Tomlinson I and J library against receptor binding domain (RBD) of SARS CoV2, eight clones which showed positive binding in phage ELISA, contained one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences. The presence of amber stop codons within the antibody sequence causes the premature termination of soluble form of scFv expression in nonsuppressor Escherichia coli strain. In the present study, we have used a novel strategy that allows soluble expression of scFvs having amber stop codon in their gene sequences (without phage PIII protein fusion), in the suppressor strain. This strategy of introduction of Ochre (TAA) codon at the junction of scFv and PIII gene, speeds up the initial screening process which is critical for selecting the right scFvs for further studies. Present strategy leads to the identification of a scFv, B8 that binds specifically with nanomolar affinity toward SARS CoV 2 RBD, which otherwise lost in terms of traditional methodology.     

Generation and characterization of human monoclonal antibodies to G5, a linear neutralization epitope on glycoprotein of rabies virus, by phage display technology

The aim of the present study was to discover distinct human MAbs to RV with high neutralizing potency and a broad neutralization spectrum. A phage display technology was used to produce human scFv to G5, a conserved linear neutralization epitope on Gp of RV. A phage display scFv library with 6 x 10(7) members was constructed and the phage-scFv with 'antigen-binding' activities were selected with synthetic peptide G5-24. The obtained scFv genes were cloned into pET22b(+)/BL21(DE3) and from this we prepared purified scFv fragments. The assay of the specificity characteristics and neutralization capacity showed that two distinct clones with new human immunoglobulin V genes can recognize G5 specifically as well as neutralize different RV strains. They have potential for inclusion in an antibodies combination aimed for use in rabies PEP.     

Bacterial expression and characterization of a novel human anti-IgE scFv fragment

Antibodies highly specific to human immunoglobulin (Ig) E are capable of selectively blocking the IgE interaction or eliminating IgE-producing cells, thus providing valuable agents for diagnostics and treatment of various allergic illness. An example is omalizumab, a humanized monoclonal anti-IgE antibody that is approved for the treatment of patients with moderate-to-severe allergic diseases in the US, European Union and other countries. Here, we describe the generation and characterization of a novel human anti-IgE as a single-chain antibody fragment (scFv). The bacterially-synthesized scFv showed high affinity (86 nM) and specificity to the Fc region of human IgE. To our knowledge, this is the first report of the production of a human anti-IgE scFv in E. coli. Its further development as a potential candidate for medical applications is discussed.Key words: anti-IgE, E. coli expression, scFv, antibody engineering, human antibodies, allergy diseases, antibody therapeutics     

Development and Implementation of a Single-Chain Fv Antibody for Specific Detection of Bacillus anthracis Spores

A single-chain Fv (scFv) antibody was developed and applied for efficient and specific detection of Bacillus anthracis spores. The antibody was isolated from a phage display library prepared from spleens of mice immunized with a water-soluble extract of the outer membrane of the B. anthracis spore (exosporium). The library (7 × 10⁶ PFU) was biopanned against live, native B. anthracis ATCC Δ14185 spores suspended in solution, resulting in the isolation of a unique soluble scFv antibody. The antibody was affinity purified and its affinity constant (3 × 10⁸ ± 1 × 10⁸ M⁻¹) determined via flow cytometry (FCM). Preliminary characterization of scFv specificity indicated that the scFv antibody does not cross-react with representatives of some phylogenetically related Bacillus spores. The potential use of scFv antibodies in detection platforms was demonstrated by the successful application of the soluble purified scFv antibody in enzyme-linked immunosorbent assays, immunofluorescence assays, and FCM.     

Engineering of a broad-specificity antibody: detection of eight fluoroquinolone antibiotics simultaneously

Recombinant sarafloxacin-recognizing antibody was engineered with the use of novel fluoroquinolone (FQ) derivatives. A monoclonal FQ antibody, 6H7, was targeted to random mutagenesis to broaden the specificity of the antibody in development of a generic assay for FQ antibiotics. Engineering involved the synthesis of different small-sized FQ molecules to immobilize and detect the mutant antibodies. Selections with labeled FQs resulted in several mutant antibodies with increased affinity or wider specificity toward different FQs. The best characterized mutant antibody was capable of recognizing seven of eight targeted FQs below maximum residue limits set by the European Union. The results are promising in regard to the development of a multiresidue immunoassay for FQs based on a single antibody.     

Preparation of antibodies against a novel immunosuppressant, FTY720, and development of an enzyme immunoassay for FTY720

FTY720 (1) is a novel immunosuppressant (immunomodulator), derived from ISP-I (2: myriocin and thermozymocidin). To clarify the pharmacokinetic properties of 1, antibodies against 1 were prepared and a competitive enzyme immunoassay (EIA) was developed. Two kinds of haptens, 3 and 4, for 1 were synthesized and coupled to ovalbumin (OVA). Rabbits were immunized with 3-OVA or 4-OVA, and corresponding antibodies were obtained. Both antibodies recognized the 2-amino-2-(2-phenylethyl)propane-1,3-diol moiety in 1. Using the anti-3-OVA antibody, a competitive EIA for 1 was developed and evaluated. The range of quantification by the EIA was 0.06-10 ng/mL. The application of the EIA has enabled us to measure the FTY720 concentration in serum after oral administration of 1 (1mg/kg) to rats.     

Use of high-capacity surface with oriented recombinant antibody fragments in a 5-min immunoassay for thyroid-stimulating hormone

High-capacity surfaces can enhance analyte-binding kinetics and be beneficial for rapid immunoassays. Site-specifically immobilized, oriented recombinant single-chain Fv (scFv) and Fab antibody fragments were compared with a conventional, nonoriented monoclonal antibody (Mab) to capture antigen from serum to solid surface in a one-step, two-site thyroid-stimulating hormone (TSH) immunoassay with a 5-min incubation time. The assay used a ready-to-use dry reagent-based concept and time-resolved fluorescent measurement. TSH binding capacities were 3.0-fold (Fab) and at least 4.1-fold (scFv) higher when recombinant antibodies were used instead of Mab. Recombinant antibody fragments also produced faster kinetics (5 vs. 45-min saturation level) than Mab: 21-25% (Mab) versus 72-83% (scFv and Fab). Analytical sensitivities of the 5-min assay were 0.09 mIU/L TSH (Fab), 0.16 mIU/L TSH (scFv), and 0.26 mIU/L TSH (Mab). Between-run variabilities were 4.2-7.9% (Fab), 4.6-17.7% (scFv), and 5.5-7.2% (Mab). The assays correlated well with the AutoDELFIA hTSH (human TSH) Ultra assay (r = 0.99, n = 109). Fab was good in all aspects of immunoassay—capacity, kinetics, sensitivity, and analytical performance. As a homogeneous, stable, and small-sized binding molecule with optimized surface-coating properties as well as reduced risk for interference by heterophilic antibodies, Fab fragment is a promising and realistic immunoreagent for the future.     

Paracrine inhibition of prion propagation by anti-PrP single-chain Fv miniantibodies

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C). A growing body of evidence suggests that antibodies to PrP(C) can antagonize deposition of PrP(Sc). However, host tolerance hampers the induction of immune responses to PrP(C), and cross-linking of PrP(C) by bivalent anti-PrP antibodies is neurotoxic. In order to obviate these problems, we explored the antiprion potential of recombinant single-chain antibody (scFv) fragments. scFv fragments derived from monoclonal anti-PrP antibody 6H4, flagged with c-myc and His6 tags, were correctly processed and secreted by mammalian RD-4 rhabdomyosarcoma cells. When cocultured with cells secreting anti-PrP scFv, chronically prion-infected neuroblastoma cells ceased to produce PrP(Sc), even if antibody-producing cells were physically separated from target cells in transwell cultures. Expression of scFv with irrelevant specificity, or of similarly tagged molecules, was not curative. Therefore, eukaryotically expressed scFv exerts a paracrine antiprion activity. The effector functions encoded by immunoglobulin constant domains are unnecessary for this effect. Because of their small size and their monovalent binding, scFv fragments may represent candidates for gene transfer-based immunotherapy of prion diseases.     

Construction and characterization of a pseudo-immune human antibody library using yeast surface display

Lymphocytes from eight individuals out of 60 healthy donors, whose plasmas showed relatively higher antibody titer for a target antigen of death receptor 5 (DR5), were selected for the source of antibody genes to construct so called an anti-DR5 pseudo-immune human single-chain fragment variable (scFv) library on the yeast cell surface (approximately 2x10(6) diversity). Compared with a large nonimmune human scFv library (approximately 1x10(9) diversity), the repertoire of the pseudo-immune scFv library was significantly biased toward the target antigen, which facilitated rapid enrichments of the target-specific high affinity scFvs during selections by fluorescence activated cell sortings. Isolated scFvs, HW5 and HW6, from the pseudo-immune library showed much higher specificity and affinity for the targeted antigen than those from the nonimmune library. Our results suggest that a pseudo-immune antibody library is very efficient to isolate target-specific high affinity antibody from a relatively small sized library.     

Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris

Anti-ErbB2 antibodies are used as convenient tools in exploration of ErbB2 functional mechanisms and in treatment of ErbB2-overexpressing tumors. When we employed the yeast Pichia pastoris to express an anti-ErbB2 single-chain antibody (scFv) derived from the tumor-inhibitory monoclonal antibody A21, the yield did not exceed 1-2 mg/L in shake flask cultures. As we considered that the poor codon usage bias may be one limiting factor leading to the inefficient translation and scFv production, we designed and synthesized the full-length scFv gene by choosing the P. pastoris preferred codons while keeping the G+C content at relatively low level. Codon optimization increased the scFv expression level 3- to 5-fold and up to 6-10 mg/L. Northern blotting further confirmed that the increase of scFv expression was mainly due to the enhancement of translation efficiency. Investigation of culture conditions revealed that the maximal cell growth and scFv expression were achieved at pH 6.5-7.0 with 2% casamino acids after 72 h methanol induction. Secreted scFv was easily purified (>95% homogeneous product) from culture supernatants in one step by using Ni2+ chelating affinity chromatography. The yield was approximately 10-15 mg/L. Functional studies showed that the A21 scFv could be internalized with high efficiency after binding to the ErbB2-overexpressing cells, suggesting this regent may prove especially useful for ErbB2-targeted immunotherapy.     

In vitro characterization of a recombinant 32P-phosphorylated anti-(carcinoembryonic antigen) single-chain antibody

 The major limitations of monoclonal antibody conjugates as therapeutic agents have been their poor tumour targeting, inadequate tumour penetration and immunogenicity. More even and deeper tissue penetration has been demonstrated with smaller antibody fragments. The smaller size and absence of an Fc segment may contribute to a lowered immunogenicity with single-chain antibodies (scFv) and also permit their recombinant engineering and bacterial expression. We describe the successful engineering, expression and pre-clinical characterisation of a phosphorylatable “kemptide” (Leu-Arg-Arg-Ala-Ser-Gly) anti-carcinoembryonic antigen (anti-CEA) scFv (PKS-scFv), for use as a radioimmunotherapeutic agent. Specifically, a yield of 6 mg/l induced culture was obtained. Site-specific phosphorylation was demonstrated without loss of specificity. In vitro assays revealed a selective cytotoxicity of ³²P-PKS-scFv for high-CEA-expressing LS-174T cells compared to the low-CEA-expressing HT-29 cells, with a rapid internalisation rate. Received: 20 March 1997 / Accepted: 5 February 1998     

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain antibodies

Since phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties, we employed phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as carbohydrate antigens. An accompanying paper in this issue describes how phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues were isolated and characterized. In this study, four independent scFv genes, isolated by a mannotriose (Man3)-bearing lipid as an antigen as previously described, were used to construct expression vectors to produce soluble scFv proteins in quantity. Both bacterial and mammalian expression systems were used to produce glutathione S-transferase-scFv fusion proteins and scFv-human IgG1 Fc conjugates, respectively. The expressed scFv fusion proteins were purified to apparent homogeneity with yields of approximately 1 and 48 mg, from 1 L of bacterial culture and myeloma cell media, respectively. Surface plasmon resonance and ELISA analyses confirmed that purified scFv proteins showed Man3 specificity. The humanized antibody in scFv-Fc form, derived from clone 5A3, was a disulfide-liked dimer with a molecular mass of 108 kDa. According to a bivalent model, the kinetics parameters of its binding to Man3 were determined to be ka = 4.03 x 104 M-1 s-1, kd = 5.77 x 10-4 s-1, KA = 6.98 x 107 M-1, and KD = 1.43 x 10-8 M. This study thus established the foundation for isolation of carbohydrate-specific scFv genes and eventual production of humanized scFv-Fc type antibodies.