To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.
Animals ; Antibodies, Bispecific ; biosynthesis ; immunology ; Antibodies, Neutralizing ; biosynthesis ; immunology ; Escherichia coli ; Fibronectins ; chemistry ; immunology ; Humans ; Mice ; Single-Chain Antibodies ; biosynthesis ; immunology ; Tumor Necrosis Factor-alpha ; immunology

Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy. To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains. The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host. After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting. The yield of scDb protein was 570 microg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR. In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.
Antibodies, Bispecific ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Humans ; Plasmids ; Protein Binding ; Receptor, Epidermal Growth Factor ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.
Base Sequence ; Cloning, Molecular ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Genetic Vectors/genetics ; Hep G2 Cells ; K562 Cells ; Molecular Sequence Data ; Receptors, Transferrin/*immunology ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/genetics ; Single-Chain Antibodies/*biosynthesis ; Single-Chain Antibodies/genetics

Secretory anti-gp96 scFv fragment was expressed in Pichia pastoris to obtain a small molecule antibody that specifically recognizes heat shock protein gp96. The gp96-scFv fragment gene was synthesized and cloned to Pichia pastoris expression plasmid pPICZa-A. Pichia pastoris X33 was electroporated with the linearized recombinant expression vector, and expression of gp96-scFv fragment was induced by methanol. The His-tagged recombinant protein was then purified by affinity chromatography and analyzed with SDS-PAGE and Western blotting assays. The biological activities of recombinant gp96-scFv fragment were determined by Western blotting, Immunofluorescence, ELISA and FACS assays. The gp96-scFv fragment was expressed successfully in Pichia pastoris. About 50 mg of recombinant protein could be purified from 1 liter of the Pichia pastoris culture supernatant. Its molecular weight was about 15 kDa. The gp96-scFv fragment could specifically bind to gp96 protein by Western blotting, immunofluorescence, ELISA and FACS analyses. Pichia pastoris-expressed gp96-scFv fragment specifically recognizes gp96 protein, which could be used for Western blotting, Immunofluorescence, ELISA and FACS analyses.
Blotting, Western ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Membrane Glycoproteins ; immunology ; Pichia ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; Single-Chain Antibodies ; biosynthesis

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.
Erythrocytes ; immunology ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

OBJECTIVETo construct a human phage antibody library and screen the single chain variable fragment (ScFv) antibudies to peroxiredoxin I (Prx I) of lung adenocarcinoma.

METHODSThe total RNA was isolated from the lymph nodes of lung cancer patients to amplify V(H) and V(L) genes by RT-PCR. V(H) and V(L) were linked with a DNA linker by SOE-PCR to construct the single chain variable fragment gene. The ScFvs were coloned into the phage vector pCANTAB5E. The insert ratio of the ScFv antibody library was identified by PCR, and the products were digested by SfiI/NotI and analyzed with 1% agarose gel electrophoresis. Three rounds of panning against lung adenocarcinoma cell line A549 and Prx I were performed, and the positive clones were identified for soluble expression. The soluble antibodies were identified by SDS-PAGE and Western blotting, and ELISA and immunocytochemistry were used to characterize the activity of the antibodies.

RESULTSA recombination phage antibody library was constructed. The insert ratio of ScFv gene was 77% (23/30), and enzyme digestion identified the target product. The sixth phage harvest resulted in a yield 180 folds of that of the first one. Positive reactions to A549 cells were detected in 6 of 10 random clones, with a positivity rate of 60%. The soluble human ScFvs against Prx I of lung adenocarcinoma were expressed in E. coli HB2151 and confirmed by SDS-PAGE and Western blotting. ELISA and immunocytochemistry demonstrated a relative specific affinity of the soluble antibodies to A549 cells.

CONCLUSIONScFv antibodies against lung adenocarcinoma have been acquired by phage display antibody library technique, and the soluble antibodies have a relative avidity specific to human lung adenocarcinoma A549 cells overexpressing PrxI.

Adenocarcinoma ; immunology ; pathology ; Antibodies, Neoplasm ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; Cell Line, Tumor ; Humans ; Immunoglobulin Variable Region ; immunology ; Lung Neoplasms ; immunology ; pathology ; Peptide Library ; Peroxiredoxins ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; immunology

OBJECTIVETo isolate single chain antibody fragments (scFv) against cell surface molecules by pathfinder selection from an anti-KG1a cell scFv phage library.

METHODSThe anti-KG1a scFv library was enriched by KGla cell panning for three rounds, or unenriched, then processed for pathfinder selection respectively using anti-CD34 monoclonal antibody as pathfinder molecule. ScFv phage clones were randomly picked and identified by binding KG1a cells using immunofluorescein and flow cytometry. The KG1a+ clones were further identified by KG1a, HL60, U937, and CEM cell lines and ELISA. Their antigenic molecules on cell surface were digested by chymopapain and analyzed by flow cytometry. DNAs from ten positive clones were sequenced. The scFv clones with different primary structure were used to analyze the molecular weight of their antigens by Western blot.

RESULTSOne hundred and two KG1a+ scFv phage clones were isolated from 144 enriched and 96 unenriched scFv phage library respectively, among which 47 bound KG1a, HL60, U937, and CEM cells, 55 bound KG1a cells exclusively. None of 28 KG1a+, HL60-, U937-, and CEM- scFv clones bound to the CD34 antigen, as confirmed by ELISA, although most of their antigens were sensitive to chymopapain digestion. DNA sequences from ten positive clones showed that they were from four different clones. They bound antigens with different molecular weight.

CONCLUSIONSOne hundred and two scFv phage clones specific for hematopoietic stem and progenitor cells have been isolated from an anti-KG1a cell scFv phage library. The pathfinder selection has showed advantages to improve the screening efficacy of scFv phage clones against antigens, which present at very low densities on the cell surface.

Antibodies, Anti-Idiotypic ; immunology ; Antibodies, Monoclonal ; genetics ; Antibody Specificity ; Bacteriophages ; genetics ; Cloning, Molecular ; Hematopoietic Stem Cells ; immunology ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Peptide Library ; Single-Chain Antibodies

We developed a new method for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin. The scFv antibody F5 could bind zebrafish vitellogenin specifically in phage-displayed form but not soluble form. The gene of scFv antibody F5 was cloned into vector pET 32a and transferred into Escherichia coli ori DE3. With inducible expression, soluble scFv antibody 32a-F5 was obtained successfully and could also specifically bind to zebrafish vitellogenin. The insoluble expression of phage-displayed scFv antibody was a common problem in the practical use of phage display. This study offered a feasible way to express soluble scFv antibodies with biological activity.
Amino Acid Sequence ; Animals ; Antibody Specificity ; Base Sequence ; Cell Surface Display Techniques ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; immunology ; Solubility ; Vitellogenins ; immunology ; Zebrafish ; immunology ; metabolism

Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Neoplasm ; biosynthesis ; immunology ; Antibody Affinity ; genetics ; Bacteriophages ; immunology ; Base Sequence ; Cell Line, Tumor ; Cells, Cultured ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Immunohistochemistry ; Liver Neoplasms ; immunology ; therapy ; Plasmids ; genetics ; Single-Chain Antibodies ; chemistry ; immunology

In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; biosynthesis ; genetics ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Mice ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; genetics ; metabolism