Thursday, April 14, 2011
Figure 1. Identifying key mutations that decrease immunogenicity. Immunogenicity needs
to be considered when designing therapeutic proteins and peptides. Immunogenic
molecules can stimulate the production of antibodies that block their
therapeutic effect or even trigger a harmful immune response.
Researchers from the National Cancer
Institute report in the Proceedings of the National
Academy of Sciences a systematic approach for identifying the B cell
epitopes in domain III of the Pseudomonas exotoxin A and point mutations
in these regions that decrease the binding of Pseudomonas exotoxin A-specific
First, researchers generate a panel of mAbs and select
for those that bind to domain III using immune complex capture ELISA [a].
Next, researchers generate a set of domain III mutants, in which large,
surface-exposed amino acid residues are substituted with smaller ones [b].
In a second ELISA step, researchers load individual
wells of a microtiter plate with a domain III mutant and mAb such that every
mutant is tested for reactivity with every mAb [c]. A secondary antibody
is added to generate a signal that measures the degree of protein-antibody reactivity
[d]. This step is used to identify key mutations that reduce the binding
of one or more mAbs by over 90% compared to a domain III mutant with a control
Researchers then use the information obtained from the
previous step to deduce the sequence of a domain III mutant containing a set of
mutations that should decrease reactivity to most, if not all, the tested mAbs.