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 mAbs.

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 mutation.

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.