Tetravalent miniantibodies with high avidity assembling in Escherichia coli.

We have designed tetravelent miniantibodies assembling in the periplasm of Escherichia coli. They are based on single-chain Fv fragments, connected via a flexible hinge to an amphipathic helix which tetramerizes the molecule. The amphipathic helix is derived from the coiled coil helix of the transcription factor GCN4, in which all hydrophobic a positions of every heptad repeat have been exchanged to leucine and all d positions to isoleucine. Gel filtration shows tetramer assembly of the miniantibody even at low concentrations. As expected, the functional affinity (avidity) of the tetravalent miniantibody is higher in ELISA and BIAcore measurements than that of the bivalent construct and the gain is dependent on surface epitope density.

[1]  D M Crothers,et al.  The influence of polyvalency on the binding properties of antibodies. , 1972, Immunochemistry.

[2]  A. Plückthun,et al.  Improved Bivalent Miniantibodies, with Identical Avidity as Whole Antibodies, Produced by High Cell Density Fermentation of Escherichia coli , 1993, Nature Biotechnology.

[3]  A. Lupas,et al.  Predicting coiled coils from protein sequences , 1991, Science.

[4]  B. Chesebro,et al.  Modification of Immunoglobulin Combining Sites , 1971 .

[5]  P. S. Kim,et al.  X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. , 1991, Science.

[6]  D Eisenberg,et al.  Crystal structure of a synthetic triple-stranded alpha-helical bundle. , 1993, Science.

[7]  A. Plückthun,et al.  Engineered turns of a recombinant antibody improve its in vivo folding. , 1995, Protein engineering.

[8]  R K Jain,et al.  Effect of bivalent interaction upon apparent antibody affinity: experimental confirmation of theory using fluorescence photobleaching and implications for antibody binding assays. , 1992, Cancer research.

[9]  R. Hodges,et al.  Packing and hydrophobicity effects on protein folding and stability: Effects of β‐branched amino acids, valine and isoleucine, on the formation and stability of two‐stranded α‐helical coiled coils/leucine zippers , 1993, Protein science : a publication of the Protein Society.

[10]  U. Jönsson,et al.  Real-time biospecific interaction analysis , 1992 .

[11]  R. Glockshuber,et al.  A comparison of strategies to stabilize immunoglobulin Fv-fragments. , 1990, Biochemistry.

[12]  Y. Kurosawa,et al.  Development of an artificial antibody system with multiple valency using an Fv fragment fused to a fragment of protein A. , 1993, The Journal of biological chemistry.

[13]  R. Karlsson,et al.  Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. , 1991, BioTechniques.

[14]  A. Plückthun,et al.  Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli. , 1992, Biochemistry.

[15]  E. Goetzl,et al.  Affinity labeling of a mouse myeloma protein which binds nitrophenyl ligands. Kinetics of labeling and isolation of a labeled peptide. , 1970, Biochemistry.

[16]  R. Karlsson,et al.  Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. , 1991, Journal of immunological methods.

[17]  L. Hood,et al.  The generation of diversity in phosphorylcholine-binding antibodies. , 1984, Advances in immunology.

[18]  P. S. Kim,et al.  A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. , 1993, Science.

[19]  W. DeGrado,et al.  Design of a 4-helix bundle protein: synthesis of peptides which self-associate into a helical protein , 1987 .

[20]  J. Richards,et al.  Structure-function relations in phosphorylcholine-binding mouse myeloma proteins. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Y. Satow,et al.  Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A. , 1985, Journal of molecular biology.