Production of Site-Specific Antibody–Drug Conjugates Using Optimized Non-Natural Amino Acids in a Cell-Free Expression System

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 in vitro cell cytotoxicity assays.

Highly accelerated inverse electron-demand cycloaddition of electron-deficient azides with aliphatic cyclooctynes

“…It must be noted, however, that the absolute rate constants as measured by this protocol are about twofold lower than those reported earlier in other solvent systems 1 . One sensible explanation lies in the fact that 1,3-dipolar cycloadditions generally proceed faster in a more polar environment (for example, 50% aqueous CH 3 CN) 23 , as for example was also demonstrated by us for strain-promoted nitrone cycloadditions 24 19,20 , reaction with BCN unexpectedly proceeded 2.9 Â faster in comparison with benzyl azide (A, entry 1). Thus, in a side-by-side comparison, reaction of phenyl azide with BCN is in fact 6 Â faster than with DIBAC, quite opposite to aliphatic azides.…”

“…Our investigations on the structure-reactivity relationship of azides in strain-promoted cycloadditions were stimulated by the fact that much effort has been devoted to the development of more reactive cyclooctynes 8,[10][11][12][13][14][15] , but only scant information is available on the influence of the azide structure on SPAAC rates 19,20 . In addition, it struck us that SPAAC reaction rate constants are nearly always determined with an aliphatic azide (typically benzyl azide), but rarely with an aromatic azide.…”

“…In addition, it struck us that SPAAC reaction rate constants are nearly always determined with an aliphatic azide (typically benzyl azide), but rarely with an aromatic azide. The reason to generally avoid aromatic azides for SPAAC presumably lies in the reported sevenfold reduced reactivity of phenyl azide in comparison with an aliphatic azide, at least in conjunction with DIBAC 19 . The observation that reaction rates of aromatic azides are hardly influenced by changing the electronic nature of substituents (as determined for p-methoxy and p-CF 3 -phenyl azide) 20 , has provided further ground to avoid aromatic azides for SPAAC.…”

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

“…46,47 Without the limitation of cell viability imposed by conventional expression systems, Xpress CF is capable of protein expression at g/L scale within hours, and has been demonstrated to retain high productivity in transition from micro-to manufacturing scale. Its open and flexible nature can tolerate multifarious manipulations in extract, DNA template, and chemical and protein additives, 46,48,49 which makes it amenable for high-throughput screening by automation. 50 Over the years, its utilization has expanded to include production of IgGs and antibody derivatives, 46 antibody-drug conjugates, 49 vaccines, 51 membrane proteins, 52 metalloproteins, 53 and viral proteins.…”

“…Its open and flexible nature can tolerate multifarious manipulations in extract, DNA template, and chemical and protein additives, 46,48,49 which makes it amenable for high-throughput screening by automation. 50 Over the years, its utilization has expanded to include production of IgGs and antibody derivatives, 46 antibody-drug conjugates, 49 vaccines, 51 membrane proteins, 52 metalloproteins, 53 and viral proteins. 54 We report bispecific antibody expression, in particular KIH, using Xpress CF in this study.…”