Development of a high yielding expression platform for the introduction of non-natural amino acids in protein sequences

Abstract

The ability to genetically encode non-natural amino acids (nnAAs) into proteins offers an expanded tool set for protein engineering. nnAAs containing unique functional moieties have enabled the study of post-translational modifications, protein interactions, and protein folding. In addition, nnAAs have been developed that enable a variety of biorthogonal conjugation chemistries that allow precise and efficient protein conjugations. These are being studied to create the next generation of antibody-drug conjugates with improved efficacy, potency, and stability for the treatment of cancer. However, the efficiency of nnAA incorporation, and the productive yields of cell-based expression systems, have limited the utility and widespread use of this technology. We developed a process to isolate stable cell lines expressing a pyrrolysyl-tRNA synthetase/tRNApyl pair capable of efficient nnAA incorporation. Two different platform cell lines generated by these methods were used to produce IgG-expressing cell lines with normalized antibody titers of 3 g/L using continuous perfusion. We show that the antibodies produced by these platform cells contain the nnAA functionality that enables facile conjugations. Characterization of these highly active and robust platform hosts identified key parameters that affect nnAA incorporation efficiency. These highly efficient host platforms may help overcome the expression challenges that have impeded the developability of this technology for manufacturing proteins with nnAAs and represents an important step in expanding its utility.

Site-selective modification strategies in antibody–drug conjugates

“…CHO cells in particular are routinely used for recombinant antibody production, including those engineered to contain ncAAs for ADC generation. [166][167][168] However, unlike bacterial expression systems mammalian cells require prolonged amber suppression, which can be damaging to cells. 167 Thus, as an alternative approach, cell-free protein synthesis (CFPS) systems have been developed.…”

“…[166][167][168] However, unlike bacterial expression systems mammalian cells require prolonged amber suppression, which can be damaging to cells. 167 Thus, as an alternative approach, cell-free protein synthesis (CFPS) systems have been developed. These systems lack the cell membrane, enabling unrestricted ncAA access to the engineered RS enzymes.…”

“…173 Indeed, ncAA position is typically optimised for each ncAA, antibody, and payload to ensure the generation of ADCs capable of efficient antigen binding, without impacting functional domains that control pharmacokinetics and stability. 167,174,175 For example, VanBrunt et al used a crystal structure of an IgG1 to select four conjugation sites that were distal to the antigen binding sites and avoided the hinge region, as well as residues known to be critical for Fc receptor binding. 175 In addition, the selected sites were predicted to be solvent exposed and outwardly oriented to enable efficient conjugation.…”

“…11E). 167,175 In one example, an anti-HER2 antibody expressing AzK on each heavy chain was conjugated to DBCO-tubulysin to give an ADC with a DAR B 2. In vitro assays demonstrated the potent and selective cytotoxicity of the resulting ADC.…”

“…In vitro assays demonstrated the potent and selective cytotoxicity of the resulting ADC. 167 2.4.6 Conjugation to ncAAs via Diels-Alder cycloadditions. As an alternative to ketone-and azide-containing ncAAs, Koehler et al have reported the incorporation of several highly reactive ncAAs based on propargyl-lysine (PrK), transcyclooctene-lysine (TCOK), cyclooctyne-lysine (SCOK) and BCNlysine (BCNK) into antibodies.…”