Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system.

Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.

[1]  P G Schultz,et al.  Expanding the Genetic Code of Escherichia coli , 2001, Science.

[2]  P. Schultz,et al.  Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base, amber, and opal suppression. , 2003, Biochemistry.

[3]  P. Schultz,et al.  A genetically encoded infrared probe. , 2006, Journal of the American Chemical Society.

[4]  J. Chin,et al.  Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA Synthetase/tRNA(CUA) pair and click chemistry. , 2009, Journal of the American Chemical Society.

[5]  John P Leonard,et al.  Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. , 2010, The New England journal of medicine.

[6]  Peter G Schultz,et al.  An Expanded Eukaryotic Genetic Code , 2003, Science.

[7]  Carolyn R Bertozzi,et al.  Cu-free click cycloaddition reactions in chemical biology. , 2010, Chemical Society reviews.

[8]  Peter G Schultz,et al.  A genetically encoded photocaged amino acid. , 2004, Journal of the American Chemical Society.

[9]  Damon L. Meyer,et al.  Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. , 2006, Bioconjugate chemistry.

[10]  P. Schultz,et al.  Genetic incorporation of a metal-ion chelating amino acid into proteins as a biophysical probe. , 2009, Journal of the American Chemical Society.

[11]  P. Schultz,et al.  An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli. , 2003, Nucleic acids research.

[12]  P. Schultz,et al.  The incorporation of a photoisomerizable amino acid into proteins in E. coli. , 2006, Journal of the American Chemical Society.

[13]  M. Sliwkowski,et al.  Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates , 2012, Nature Biotechnology.

[14]  P. Schultz,et al.  Addition of the keto functional group to the genetic code of Escherichia coli , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Carolyn R. Bertozzi,et al.  Copper-free click chemistry for dynamic in vivo imaging , 2007, Proceedings of the National Academy of Sciences.

[16]  M. Dorywalska,et al.  Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. , 2013, Chemistry & biology.

[17]  S. Brewer,et al.  Sensitive, site-specific, and stable vibrational probe of local protein environments: 4-azidomethyl-L-phenylalanine. , 2013, The journal of physical chemistry. B.

[18]  John M Lambert,et al.  Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. , 2008, Cancer research.

[19]  N. Dixon,et al.  High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites. , 2012, Biochemical and biophysical research communications.

[20]  P. Schultz,et al.  Adding L-3-(2-Naphthyl)alanine to the genetic code of E. coli. , 2002, Journal of the American Chemical Society.

[21]  C. J. Murray,et al.  Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system , 2012, mAbs.

[22]  R. Lutz,et al.  Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. , 2006, Cancer research.

[23]  Ryohei Ishii,et al.  Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. , 2008, Chemistry & biology.

[24]  M. Wolfert,et al.  Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast huisgen cycloadditions. , 2008, Angewandte Chemie.

[25]  C. J. Murray,et al.  Microscale to Manufacturing Scale-up of Cell-Free Cytokine Production—A New Approach for Shortening Protein Production Development Timelines , 2011, Biotechnology and bioengineering.

[26]  Peter G Schultz,et al.  Synthesis of site-specific antibody-drug conjugates using unnatural amino acids , 2012, Proceedings of the National Academy of Sciences.

[27]  Adam C. Fisher,et al.  Laboratory Evolution of Fast-Folding Green Fluorescent Protein Using Secretory Pathway Quality Control , 2008, PloS one.

[28]  Thomas Huber,et al.  Multiple-site labeling of proteins with unnatural amino acids. , 2012, Angewandte Chemie.

[29]  P. Schultz,et al.  The genetic incorporation of a distance probe into proteins in Escherichia coli. , 2006, Journal of the American Chemical Society.

[30]  Peter G Schultz,et al.  An enhanced system for unnatural amino acid mutagenesis in E. coli. , 2010, Journal of molecular biology.

[31]  J. Baselga,et al.  Trastuzumab emtansine for HER2-positive advanced breast cancer. , 2012, The New England journal of medicine.

[32]  Andrew B. Martin,et al.  Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. , 2002, Journal of the American Chemical Society.

[33]  Paul Polakis,et al.  Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index , 2008, Nature Biotechnology.

[34]  P. Schultz,et al.  Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon* , 2010, The Journal of Biological Chemistry.

[35]  James F Zawada.  Preparation and testing of E. coli S30 in vitro transcription translation extracts. , 2012, Methods in molecular biology.

[36]  James R. Swartz,et al.  High‐level cell‐free synthesis yields of proteins containing site‐specific non‐natural amino acids , 2009, Biotechnology and bioengineering.

[37]  P. Burke,et al.  A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology. , 2013, Bioconjugate chemistry.

[38]  K. Grabstein,et al.  Development of copper-catalyzed azide-alkyne cycloaddition for increased in vivo efficacy of interferon β-1b by site-specific PEGylation. , 2012, Bioconjugate chemistry.

[39]  C. Wood,et al.  Potent Anticarcinoma Activity of the Humanized Anti-CD70 Antibody h1F6 Conjugated to the Tubulin Inhibitor Auristatin via an Uncleavable Linker , 2008, Clinical Cancer Research.

[40]  B. E. Kimmel,et al.  Optimized clinical performance of growth hormone with an expanded genetic code , 2011, Proceedings of the National Academy of Sciences.

[41]  Peter G. Schultz,et al.  Expanding the genetic code. , 2006 .

[42]  Susan E. Cellitti,et al.  Site-specific labeling of proteins with NMR-active unnatural amino acids , 2010, Journal of biomolecular NMR.

[43]  P. Schultz,et al.  One plasmid selection system for the rapid evolution of aminoacyl-tRNA synthetases. , 2009, Bioorganic & medicinal chemistry letters.

[44]  P. Schultz,et al.  An efficient system for the evolution of aminoacyl-tRNA synthetase specificity , 2002, Nature Biotechnology.