Several technologies aim to provide chemically amenable sites within a protein sequence for the posttranslational chemical
conjugation of small-molecule drugs, peptides, or other constructs to improve or add functionality. For example, sequence-specific
conjugations producing homogeneous ADCs with fixed payloads are aimed at improving tumor-cell killing and increasing therapeutic
index. Engineered ThioMabs (Roche/Genentech technology) that use natural cysteine residues that must be carefully unmasked
during production for subsequent site-specific conjugation have shown preclinical proof-of-concept (2). These approaches await
further clinical validation.
Carlos Barbas' laboratory at The Scripps Research Institute exploited the use of exposed tyrosine residues within the complimentarity
determining regions (CDRs) of IgG molecules as the basis for linking drug conjugates. CovX, acquired by Pfizer in 2008, was
founded to develop this technology. These sorts of approaches are attractive in that posttranslational chemical coupling to
a common IgG construct with resulting extended half-life represents a platform amenable to many different small-molecule or
Non-natural amino acids
The introduction of non-natural amino acids (nnAAs), or those amino acids not part of the 20 naturally incorporated ones into
proteins, plays an important role in basic peptide and protein research. They are increasingly used to develop biologics with
enhanced pharmacological properties beyond providing sites for drug conjugation. NnAAs can be introduced through chemical
synthesis in peptides or biosynthetically in proteins. Currently, only peptides and very small protein drugs with nnAAs are
on the market because they can be made synthetically and avoid the limitations of cell-based expression systems. A prominent
example is the semisynthetic, broad-spectrum antibiotic, ampicillin, into which the nnAAs D-phenylglycine and D-4-hydroxyphenylglycine
have been incorporated (3).
The opportunities to broaden protein diversity and properties with nnAAs are enormous, as is the ability to incorporate chemical
modifications in proteins that can endow current biopharmaceuticals with improved or new properties. These chemical modifications
can change the characteristics of proteins, including ligand-binding properties, stability, spectroscopic properties, folding
behavior, catalytic efficiency, and substrate specificity. These modifications provide possibilities to develop biobetters
and biosuperiors that have superior pharmacological properties, including improved safety profiles, longer half-life, and
enhanced activity (4).
Much effort has been put into developing technologies that ensure a site-specific incorporation of the nnAAs with a high rate
of yield. Various methods for site-specific introduction have been established, both semisynthetic and recombinant methods.
Few methods, however, have made it from the bench at small-scale protein production to commercial scale.
A recently formed biotechnology company, Redwood Biosciences, is using an approach based on the work of Carolyn Bertozzi's
laboratory at the University of California, Berkeley. Her work focuses on genetically encoded aldehyde tags and aims to exploit
a specific sequence (originally found within the sequence of sulphatases) that is posttranslationally recognized and modified
by a formyl glycine-generating enzyme to produce a so-called aldehyde chemical handle (5). The incorporation of the CxPxR
sequence at specific positions in candidate protein therapeutics provides a means to produce a site-specific nnAA with a reactive
aldehyde amenable to drug conjugation.
One of the oldest methods for nnAA incorporation into proteins uses auxotrophic strains from E. coli that cannot synthesize a specific natural amino acid and thus have to uptake it from the growth medium. A structurally similar
nnAA can be supplied within the growth medium in place of the natural amino acid and will be alternatively incorporated into
the protein. A major downside is that the specific nnAA will be incorporated at every site coding for the natural amino acid
and can lead to misfolding and impaired function of the target protein, or the nnAA can be incorporated in the host-cell's
proteins, which can have toxic effects (6). Allozyne has pioneered this type of cell-based expression system that incorporates
nnAAs into protein sequences, but this approach requires extensive re-engineering of the target protein sequence used, due
to the region-specific nature of the nnAA incorporation using this method.
Ambrx has developed cell-based nnAA incorporation systems where E. coli or CHO cells are engineered with orthogonal pairs of transfer (tRNA) and tRNA synthetases to charge and incorporate nnAAs
at selected codons at specific points in the coding sequence of the expressed protein. This approach is a significant advance
and provides answers to at least some of the questions raised about nonspecific sites of conjugations in ADCs. For truly expanding
the number and variety of nnAA that can be incorporated to determine the effect on function, even at a single amino-acid position,
the approach demands significant investment to engineer further orthogonal pairs of tRNA synthetases and tRNAs that can recognize
a library of nnAAs. A further complication is that these pairs should be exquisitely selective over natural amino acids to
avoid their incorporation over the desired nnAA, which can be challenging in a drug-manufacturing context with strict regulatory
requirements. When this challenge is taken into account, along with the variability in efficiency with which nnAAs are absorbed
into the cell, these systems will not likely be amenable to fast reiterative make-test cycles with libraries of nnAAs at multiple
sites of incorporation.
All of these considerations suggest a clear need to move away from the conventional cell-based protein expression systems
to address the critical requirement for a rapid make-test system that is amenable to many parallel re-iterations of site-specific
incorporations of defined natural amino-acid sequences or multiple nnAAs at multiple sites. The answer may not lie with cell-based
systems at all, but with completely in vitro biochemical protein synthesis based on novel cell-free expression systems.