Tetravalent Miniantibodies with High Avidity Assembling in Escherichia coli                                                                                                    

Peter Pack, 
Kristian Mu ller, 
Ralph Zahn and 
Andreas Plu kthun 

Biochemisches Institut 
Universita t Zurich
Winterthurestr.190,CH-8057 
Zurich, Switzerland   

We have designed tetravelent miniantibodies assembling in the periplasm of       
Escherichia coli They are based on single-chain Fv fragments, connected via
a exible 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 ositions of every heptad repeat    
have been exchanged to leucine and all dpositions to isoleucine. Gel ltration   
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               
onstruct and the gain is dependent on surface epitope density.                  



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