Tetravalent Miniantibodies with High Avidity Assembling inEscherichia colo

Pack, Peter; Müller, Kristian M.; Zahn, Ralph; Plückthun, Andreas

doi:10.1006/jmbi.1994.0062

Cited by 136 publications

(87 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…3). This value is within the range expected from the transformation from monovalent to bivalent miniantibodies (28,29) and probably reflects the substantial decrease in avidity of the 6AC3 MAb after becoming a monovalent scFv fragment. No TGEV-binding was detected using the EHLYA protein and an unrelated E-tagged scFv (B4).…”

Section: Downloaded Fromsupporting

confidence: 70%

Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants ofEscherichia coliCultures by Use of the Hemolysin System

Fernández

Sola

Enjuanes

et al. 2000

Appl Environ Microbiol

View full text Add to dashboard Cite

A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants of Escherichia coli cultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) of E. coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFv's can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.The current methodology for the selection and production of antibody fragments in Escherichia coli involves the generation of large repertories of human or murine immunoglobulin (Ig) V gene segments, either from naive or immunized individuals, and its cloning in filamentous phage (or phagemid) vectors that allow both the phage display and the production of the reconstructed antibody Fv fragments (17,19,25,27). After a selection (biopanning) of Fv clones capable of binding a given antigen, the recombinant Fv antibodies are produced individually in E. coli and tested for their antigen-binding properties (16,22). The standard Ig fragments produced in E. coli are the so-called single-chain Fv (scFv) in which the variable domains from the heavy (V H ) and light (V L ) chains are linked in a single polypeptide. The standard protocol for production of scFv's require their translocation to the periplasmic space using an N-terminal signal peptide (SP) that is recognized by the general secretion pathway of E. coli, encoded by the sec genes, and which is responsible for the export of most cellular proteins targeted to the extracytoplasmic compartments (12, 31). Next, the scFv polypeptides are purified, using chromatographic techniques, from periplasmic protein extracts obtained from those cells (30).Besides being time-consuming, the major problem associated with the production of scFv in E. coli is the toxicity caused by their periplasmic export and accumulation, which eventually leads to the lysis of the bacterial cell (25, 30). The export of scFvs gives rise to a number of toxic effects, such as the jamming of the Sec pathway, the titration of periplasmic-folding catalysts, the induction of periplasmic proteases, and an enhanced outer membrane permeability (3,6,7,20,32). All of these events have important biotechnological consequences, such as low production yields and the formation of scFv aggregates. Thus, an ideal method for scFv production should allow their secretion to the extracellular space without a periplasmic intermediate and by a Sec-independent pathway.The hemolysin transport system (Hly) is a type I secretory apparatus that forms a protein channel between the inner and outer membranes of E. coli through which the hemolysin toxin (HlyA) is secreted (5). The protein machinery of Hly is independent of the cellular sec genes and consists in two inner membrane components, ...

show abstract

Section: Downloaded Fromsupporting

confidence: 70%

Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants ofEscherichia coliCultures by Use of the Hemolysin System

Fernández

Sola

Enjuanes

et al. 2000

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…For tetramerization, the multimerization peptide of the human tumor suppressor protein p53 was chosen. The tetramer assembles from two dimers that associate via hydrophobic interactions of ␣-helices and ␤-strands (15,31,53). In extending our previous studies (35).…”

Section: Construction Of Multimeric and Pegylated Miniantibodies-mentioning

confidence: 63%

“…Although available data demonstrate that they greatly vary in tumor targeting efficiency (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40), direct comparisons are problematic because of the great differences among the various tumor models and antigens used.…”

mentioning

confidence: 99%

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

Kubetzko

Balic

Waibel

et al. 2006

Journal of Biological Chemistry

Self Cite

112

View full text Add to dashboard Cite

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185 HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

show abstract

“…the generation of mini-antibodies [41,42], the assembly of receptor ectodomain subunits [9,19,[43][44][45], and as an affinity tag system for purification and biosensor applications [8][9][10][46][47][48]. Immobilized EGF has been shown to be more mitogenic for CHO cells than soluble EGF [29], perhaps due to decreased ligand inter- nalization and degradation [30].…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Lenferink

Pinard

et al. 2009

Protein Expression and Purification

View full text Add to dashboard Cite

Tetravalent Miniantibodies with High Avidity Assembling inEscherichia colo

Cited by 136 publications

References 15 publications

Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants ofEscherichia coliCultures by Use of the Hemolysin System

Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants ofEscherichia coliCultures by Use of the Hemolysin System

PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Contact Info

Product

Resources

About