Hardiman: Role of research in developing anthrax countermeasures 289 [47]. Taken together, these results may serve as the basis for future applications to target these proteins as a therapeutic approach to countering anthrax infection. e n Gin e e Rin G Th e TRAn Sl o Co n AS A Pe PTiDe De l iVe Ry Sy STe M While AT co-opts host machinery to ef- fect cellular dysfunction, researchers are seeking to co-opt the activity of AT for ben- eficial purposes. Dr. Collier’s group is com- pleting work using the finely calibrated translocation system to target and deliver proteins of therapeutic benefit to a specific subset of cells. Given the robust assembly and delivery of substrates by PA, Collier and colleagues are investigating whether they can manipulate this system to selectively tar- get and kill dysregulated human cells in var- ious disease contexts. In the final portion of his talk, Dr. Col- lier offered insights into current work in the lab focusing on engineering AT to deliver toxic proteins exclusively into specified cells. His team has fused the epidermal growth factor (EGF) protein to the previ- ously characterized C-terminus of the PA re- ceptor domain [48]. His group is investigating if this fusion can change the re- ceptor specificity of AT to bind the EGF re- ceptor (EGFR). As certain types of cancers have upregulated EGFR at the cell surface [49], AT bound to EGF would specifically target cancerous cells for intoxication. Be- cause the receptor tropism is altered, this could function in a precise manner, homing in on dysregulated cells while leaving healthy cells intact. Indeed, Dr. Collier de- scribed preliminary experiments indicating that the chimeric fusion competes with free EGF for binding to EGFR, whereas native AT does not. Future work will continue in the lab on this promising translational appli- cation, which has seen many benefits using a similar paradigm with other fusion pro- teins. If Dr. Collier is successful, he will have optimized a system he conceived in the early 1980s. He previously described a fusion pro- tein consisting of DTA fused to EGF as a technique to downregulate EGFR overex- pressing cells [50]. With this strategy, the domain containing the ADP-riboslylation activity of DT is delivered specifically to pathogenic, EGFR-overproducing cells. Subsequent work by Dr. John Murphy’s group in the 1990s saw commercial success with the replacement of the receptor binding domain of DT with interleukin-2 (IL-2) to yield chimeric DT-IL2 [51]. This fusion pro- tein operates in an analogous manner to that of Dr. Collier’s group, whereby the ADP-ri- bosylating activity of DT would intoxicate cells expressing IL-2 receptors at the sur- face. A similar fusion expressing the DT cat- alytic domain (DT389) fused to IL-2 is marketed as Ontak® (Denileukin diftitox) by Eisai Corporation, having gained full FDA approval in 2008 [52]. It is indicated for the treatment of cutaneous T-cell lym- phoma, characterized by high IL-2 receptor expression in cancerous cells. Thus, Dr. Col- lier’s work seems poised to take advantage of the promising field of toxin fusion thera- peutics as a platform for selective drug de- livery. Drug delivery is the most immediate application of repurposing toxins for thera- peutic use, but other creative approaches may provide additional platforms for mo- lecular delivery. Dr. Collier noted in the seminar that the PA pore is capable of bind- ing a broad array of peptides in addition to its native LF and EF substrates. That is, LF and EF contain a polybasic leader sequence [53] that is recognized by the pore or auxil- iary cellular elements. However, this recog- nition is promiscuous, where the PA pore can translocate substrates with multiple amino acids combinations in the leader se- quence. This is likely due to the function of the α clamp, which binds peptide helices non-specifically and could confer broad specificity to recognize multiple substrates [40]. Dr. Collier observed that LFn fused to an N-terminal His-6 tag was competent for translocation [37]. Further, an N-terminal Lys-6 sequence fused to the model non-na- tive substrate DTA was found to translocate, albeit inefficiently [54]. Thus, as leader se-