A surface expression vector for antibody screening

Breitling, F.; Dübel, Stefan; Seehaus, Thomas; Klewinghaus, I; Little, Melvyn

doi:10.1016/0378-1119(91)90244-6

Cited by 287 publications

(119 citation statements)

References 23 publications

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“…The plasmid pFv90 was a derivative of the expression vector pSEX [21]. It is inducible by IPTG and contains a procaryotic signal peptide of 26 amino acid residues allowing the secretion of the recombinant protein into the periplasm of the bacteria.…”

Section: Cloning Of Pap II Into a Procaryotic Expression Vectormentioning

confidence: 99%

Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E. coli

1994

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Three distinct ribosome-inactivating proteins (RIPS) were isolated from pokeweed (Phytolacca americana). We identified and sequenced for the first time a complete cDNA encoding the pokeweed antiviral protein II (PAP II), which is expressed in the late summer leaves of pokewecd. The cDNA of PAP II consists of 1,187 nucleotides and encodes a mature protein of 285 amino acids. Its predicted amino acid sequence is only 33% similar to PAP and PAP-S. The NH2 terminal extrapeptide (25 amino acid residues) was similar but not identical to that of PAP's extrapeptide. The cDNA of PAP II was expressed in E. coli. The growth of the transformants was strongly inhibited after induction of the gene. Furthermore, PAP II, which was produced in E. coli, inhibited protein synthesis in a rabbit reticulocyte translation system. Thus, recombinant PAP II would appear to be as functional as native PAP in inhibiting protein synthesis in both prokaryotes and eukaryotes.

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Section: Cloning Of Pap II Into a Procaryotic Expression Vectormentioning

confidence: 99%

Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E. coli

1994

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“…The ligation mixture was used to transform E. coli TG1, and clones containing the correct insert identified by PCR screening and DNA sequencing. Native diabody was expressed (Breitling, 1991) and purified from the bacterial periplasm using IMAC (Hochuli et al, 1988) followed by FPLC size-exclusion chromatography using a Superdex 75 column.…”

mentioning

confidence: 99%

Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu

Adams

Schier²,

Am³

et al. 1998

Br J Cancer

184

110

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Summary Single-chain Fv (scFv) molecules exhibit highly specific tumour-targeting properties in tumour-bearing mice. However, because of their smaller size and monovalent binding, the quantities of radiolabelled scFv retained in tumours limit their therapeutic applications. Diabodies are dimeric antibody-based molecules composed of two non-covalently associated scFv that bind to antigen in a divalent manner. In vitro, diabodies produced from the anti-HER2/neu (c-erbB-2) scFv C6.5 displayed approximately 40-fold greater affinity for HER2Ineu by surface plasmon resonance biosensor measurements and significantly prolonged association with antigen on the surface of SK-OV-3 cells (tl,2 cell surface retention of > 5 h vs 5 min) compared with C6.5 scFv. In SK-OV-3 tumour-bearing scid mice, radioiodinated C6.5 diabody displayed a highly favourable balance of quantitative tumour retention and specificity. By as early as 4 h after i.v. administration, significantly more diabody was retained in tumour (10 %ID g-1) than in blood (6.7 %ID ml-') or normal tissue (liver, 2.8 %ID g-'; lung, 7.1 %ID g-1; kidney, 5.2 %ID g-1). Over the next 20 h, the quantity present in blood and most tissues dropped approximately tenfold, while the tumour retained 6.5 %ID g-1 or about two-thirds of its 4-h value. In contrast, the 24-h tumour retention of radioiodinated C6.5 scFv monomer was only 1 %ID g-1. When diabody retentions were examined over the course of a 72-h study and cumulative area under the curve (AUC) values were determined, the resulting tumor-organ AUC ratios were found to be superior to those previously reported for other monovalent or divalent scFv molecules. In conclusion, the diabody format provides the C6.5 molecule with a distinct in vitro and in vivo targeting advantage and has promise as a delivery vehicle for therapeutic agents.

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“…The cells were fixed, permeabilized, and incubated with serum A, which recognizes the N-terminal epitope of secreted scFv on Western blots. 9 A strong fluorescence appeared to be associated with membranes of the endoplasmic reticulum surrounding the nucleus. No fluorescence was detected with untransfected SkMel63 cells (data not shown).…”

Section: Resultsmentioning

confidence: 98%

A cell surface-displayed anti-c-myc single-chain antibody: new perspectives for the genetic improvement of cellular tumor vaccines

et al. 2000

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We have shown recently that cell surface-bound, single-chain Fv antibodies (scFv) are a powerful tool for the improvement of cellular tumor vaccines. To simplify this approach and to develop a general tool for the generation and improvement of cellular tumor vaccines, we chose an scFv against a peptide from the human proto-oncogene c-myc that could anchor any c-myc-tagged protein to the cell surface. The retroviral vector p50-Mx-neo (pMESV) was used to express scFv on the surface of the human melanoma line SkMel63. The cell-bound anti-c-myc scFv bound specifically to a soluble purified anti-CD28 scFv carrying a c-myc peptide-tag at its C terminus. Proof of principle was determined by incubating human peripheral blood lymphocytes with a mixture of (a) anti-c-myc-transfected SkMel63 cells binding the anti-CD28 scFv and (b) SkMel63 cells transfected with an anti-CD3 scFv. A clear synergistic effect on T-cell activation was observed that was comparable with that obtained in previous studies using SkMel63 cells transfected with the gene for the anti-CD28 scFv. As the cell surface-displayed anti-c-myc scFv can bind any c-myc-tagged protein of interest, this technique facilitates the genetic engineering of cellular vaccines for the therapy of virtually all human neoplasias.

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A surface expression vector for antibody screening

Cited by 287 publications

References 23 publications

Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E. coli

Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E. coli

Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu

A cell surface-displayed anti-c-myc single-chain antibody: new perspectives for the genetic improvement of cellular tumor vaccines

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