Protein re-surfacing of E. coli L-Asparaginase to evade pre-existing anti-drug antibodies and hypersensitivity responses.

The optimal use of many biotherapeutics is restricted by Anti-drug antibodies (ADAs) and hypersensitivity responses which can affect potency and ability to administer a treatment. Here we demonstrate that Re-surfacing can be utilized as a generalizable approach to engineer proteins with extensive surface residue modifications in order to avoid binding by pre-existing ADAs. This technique was applied to E. coli Asparaginase (ASN) to produce functional mutants with up to 58 substitutions resulting in direct modification of 35% of surface residues. Re-surfaced ASNs exhibited significantly reduced binding to murine, rabbit and human polyclonal ADAs, with a negative correlation observed between binding and mutational distance from the native protein. Reductions in ADA binding correlated with diminished hypersensitivity responses in an in vivo mouse model. By using computational design approaches to traverse extended distances in mutational space while maintaining function, protein Re-surfacing may provide a means to generate novel or second line therapies for life-saving drugs with limited therapeutic alternatives.

Stress responses to heterologous membrane protein expression in Escherichia coli.

The stress response of E. coli to the expression of two recombinant membrane proteins, the E. coli AAA+ protease FtsH and the human G-protein coupled receptor CB1, was examined using several members of a promoter-GFP library. Several genes from the heat-shock and envelope stress regulons (rpoH, clpP, lon, and ftsH) were strongly induced by expression of either membrane protein. Flow cytometry was used to monitor the real-time dynamics of the transcription of these reporter genes in response to membrane protein expression. Co-expression of CB1 and FtsH led to an additive response in these four reporter genes suggesting that the stresses may be occurring via different physiological mechanisms.