2 research outputs found
Incorporation of a Doubly Functionalized Synthetic Amino Acid into Proteins for Creating Chemical and Light-Induced Conjugates
Z-Lysine (ZLys) is a lysine derivative
with a benzyloxycarbonyl
group linked to the ε-nitrogen. It has been genetically encoded
with the UAG stop codon, using the pair of an engineered variant of
pyrrolysyl-tRNA synthetase (PylRS) and tRNA<sup>Pyl</sup>. In the
present study, we designed a novel Z-lysine derivative (AmAzZLys),
which is doubly functionalized with amino and azido substituents at
the meta positions of the benzyl moiety, and demonstrated its applicability
for creating protein conjugates. AmAzZLys was incorporated into proteins
in Escherichia coli, by using the ZLys-specific
PylRS variant. AmAzZLys was then site-specifically incorporated into
a camelid single-domain antibody specific to the epidermal growth
factor receptor (EGFR). A one-pot reaction demonstrated that the phenyl
amine and azide were efficiently linked to the 5 kDa polyethylene
glycol and a fluorescent probe, respectively, through specific bio-orthogonal
chemistry. The antibody was then tested for the ability to form a
photo-cross-link between its phenylazide moiety and the antigen, while
the amino group on the same ring was used for chemical labeling. When
incorporated at a selected position in the antibody and exposed to
365 nm light, AmAzZLys formed a covalent bond with the EGFR ectodomain,
with the phenylamine moiety labeled fluorescently prior to the reaction.
The present results illuminated the versatility of the ZLys scaffold,
which can accommodate multiple reactive groups useful for protein
conjugation
Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes
The
site-specific chemical conjugation of proteins, following synthesis
with an expanded genetic code, promises to advance antibody-based
technologies, including antibody drug conjugation and the creation
of bispecific Fab dimers. The incorporation of non-natural amino acids
into antibodies not only guarantees site specificity but also allows
the use of bio-orthogonal chemistry. However, the efficiency of amino
acid incorporation fluctuates significantly among different sites,
thereby hampering the identification of useful conjugation sites.
In this study, we applied the codon reassignment technology to achieve
the robust and efficient synthesis of chemically functionalized antibodies
containing <i>N</i><sup>ε</sup>-(<i>o</i>-azidobenzyloxycarbonyl)-l-lysine (<i>o</i>-Az-Z-Lys)
at defined positions. This lysine derivative has a bio-orthogonally
reactive group at the end of a long side chain, enabling identification
of multiple new positions in Fab-constant domains, allowing chemical
conjugation with high efficiency. An X-ray crystallographic study
of a Fab variant with <i>o</i>-Az-Z-Lys revealed high-level
exposure of the azido group to solvent, with six of the identified
positions subsequently used to engineer “Variabodies”,
a novel antibody format allowing various connections between two Fab
molecules. Our findings indicated that some of the created Variabodies
exhibited agonistic activity in cultured cells as opposed to the antagonistic
nature of antibodies. These results showed that our approach greatly
enhanced the availability of antibodies for chemical conjugation and
might aid in the development of new therapeutic antibodies