Factors Influencing the Dimer to Monomer Transition of an Antibody Single-Chain                                                
Fv Fragment                                                               

Katja M. Arndt, Kristian M. Muller, and Andreas Pluckthun
Biochemisches Institut, UniVersita t Zurich, Winterthurer strasse190,
CH-8057Z urich, Switzerland                     



ABSTRACT:
Antibody single-chain Fv (scFv) fragments are able to form dimers under certa inconditions,                                
and the extent of dimerization appears to depend on linker length, antibody sequence, and external factors.             
We analyzed the factors influencing dimer- monomer equilibrium as well as the rate of interconversion,                  
using the scFv McPC603 as a model system. In this molecule, the stability of theHV- VL interaction can                  
be conveniently varied by adjusting the ionic strength (because of its influence on the hydrophobic effect),            
by pH (presumably because of the presence of titratable groups in the interface), and by the presence or                
absence of the antigen phosphorylcholine, which can be rapidly removed due to its very fast off-rate. It                
was found that the monomer is the thermodynamically stable form with linkers of 15 and 25 amino acids                   
length under all conditions tested (35M or less). The dimer is initially formed in periplasmic expression,             
\presumably by domain swapping, and can be trapped by all factors which stabilize theH- VVL interface,                   
such as the presence of the antigen, high ionic strength, and pH below 7.5. Under all other conditions,                 
it converts to the monomer. Predominantly monomer is obtained during in vitro folding. Monomer is                       
stabilized against dimerization at very high concentrations by the same factors which stabilize theH- V                 
VL interaction.    These results should be helpful in producing molecules with defined oligomerization                  
states.                                                                                                                 



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