Abstract
Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.
Highlights
Consistent and robust production processes for the site-specific generation of antibody-drug conjugates (ADCs) have the potential to generate therapeutic products comprised of a single molecular entity rather than the heterogeneous mixtures present in the approved products of today (Adcetris[1] and Kadcyla[2])
More recent work has shown that release factor 1 (RF1) can be removed from certain strains of E. coli, provided that RF2 expression is increased by removing the RF2 autoregulation +1 frameshift and “fixing” the A246T mutation – though this generally leads to a slower growth rate of the engineered cells[10]
In this study we demonstrate that the prfA gene of E. coli that codes for RF1 can be reengineered to code for a mutant RF1 (RF1MUT) that is sensitive to out membrane protease (OmpT) protease cleavage, allowing normal cell growth rates for highly active extract production
Summary
Consistent and robust production processes for the site-specific generation of antibody-drug conjugates (ADCs) have the potential to generate therapeutic products comprised of a single molecular entity rather than the heterogeneous mixtures present in the approved products of today (Adcetris[1] and Kadcyla[2]). As RF1 was once thought to be an essential protein, critical for cell growth[13], many indirect approaches to inactivate RF1 in cell-free expression systems were first employed, including using inhibiting antibodies[6] or RNA aptamers[7], RF1 depletion by subtractive affinity chromatography[8], or excluding RF1 from recombinantly reconstituted cell-free systems (i.e. PURE)[9]. These approaches add significant process requirements and cost to generate an RF1 attenuated cell-free protein production system. To maintain scalability of our system, it was crucial not to compromise growth rate, which is important for extract activity[15], nor to incur any additional processing steps or costly additives, such as inactivating antibodies to RF1
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