The objective of the work described in this application is to optimize existing diagnostic and therapeutic antibodies for BioDefense using directed evolution approaches. Diversa Corporation will apply its unique protein engineering tools to create antibody variants, which USAMRIID will screen for improvements using the proper containment facilities. Ultimately, a robust, highly reproducible and systematic optimization process will be developed that may deliver antibodies with disassociation constants in the femtamolar range. ? 1. Clone and express three diagnostic monoclonal antibodies in bacteria as Fab fragments. ? 2. Optimize binding characteristics of three diagnostic antibodies using directed evolution technologies. ? 3. Study antibody thermal stability using molecular evolution technologies. ? 4. Improve optimization process to reduce cost, time and effort required to create more sensitive diagnostic antibodies. ? 5. Optimize additional diagnostic and therapeutic antibodies. ? Three diagnostic antibodies, alphavirus Mab SLK42, Ricin Mab 14F11 and Yersinia pestis Mab 6H3 will be optimized for improved diagnostic properties. Each of these antibodies would benefit from improved sensitivity. Diversa's Gene Site Saturation Mutagenesis(tm) (GSSM) technology will be used to produce a large collection of variants for each antibody. These variants will be quality controlled at Diversa and then shipped to USAMRIID to be assayed for function. Analysis of the data produced by this work is expected to provide new insights about antibody engineering and will likely reduce the effort required to optimize future antibodies. ? ?
| Demarest, Stephen J; Chen, Gang; Kimmel, Bruce E et al. (2006) Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression. Protein Eng Des Sel 19:325-36 |
