An Engineered Open Cell Free Synthesis Platform for the Production of Non-natural Amino Acid Based Antibody-Drug Conjugates

We describe an open cell free synthesis (OCFS) platform that facilitates the high-level production of IgGâ??s that contain non-natural amino acid (nnAA) substitutions for the site-specific conjugation of small molecule warheads. We demonstrate that targeted proteolytic inactivation can be used as a strategy to destroy the E.coli protein Release Factor 1 (RF1), boosting Amber codon mediated nnAA incorporation into a nascent polypeptide, due to lack of competition between a truncated RF1 and an orthogonal, CUA-encoded, nnAA-charged tRNA. Several strains were generated that contain variants in the switch loop of RF1 which are susceptible to cleavage by the E.coli protease OmpT. During the cell lysis step of extract production for OCFS, OmpT, which is normally compartmentalized in the E.coli outer membrane as an integral membrane protein, is mixed with the E.coli cytoplasmic fraction, and targeted proteolysis of the variant RF1 occurs on demand. Using an extract from this designed strain for OCFS, we demonstrate that itâ??s possible to incorporate nnAAâ??s into previously unobtainable sites in an IgG, producing antibodies at titers of several hundred milligrams per liter in an overnight reaction. These variants were then site specifically conjugated to a small molecule warhead and the corresponding ADCâ??s effectively killed cells in a highly efficient manner.