Production of Site-Specific Antibody–Drug Conjugates
Using Optimized Non-Natural
Amino Acids in a Cell-Free Expression System
- Publication date
- Publisher
Abstract
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 <i>in vitro</i> cell cytotoxicity assays