Modification of immunoglobulin G (IgG) 1 proteins in cancer treatment is a rapidly growing field of research. Antibody-drug conjugates (ADCs) exploit the targeted nature of this immunotherapy by conjugating highly potent drugs to antibodies, allowing for effective transport of cargo(s) to cancerous cells. Of the many bioconjugation strategies now available for the formation of highly homogenous ADCs, disulfide modification is considered an effective, low-cost and widely accepted method for modifying IgG1s for improved clinical benefit. However, little is known about how disulfide modification impacts clinically relevant fragment crystallisable (Fc) region interactions. Although often overlooked as a secondary ADC function, Fc interactions could prove key in rational design of cancer cell-targeting ADCs through consideration of potent mechanisms such as antibodydependant cellular cytotoxicity (ADCC). This work explores different IgG1 disulfide modification techniques and the effect they have on quantifiable secondary IgG1 Fc interactions (e.g. CD16a and FcRn). The solvent accessible disulfide residues of trastuzumab, a clinically relevant IgG1, were modified to provide a range of bioconjugates with differing amounts of inter-chain covalent linkages. It was found that by natively re-bridging the IgG1 model, all tested Fc functionalities were not significantly affected. Additionally, in non Fc-specific biophysical experiments (e.g. thermal stability/aggregation), the natively re-bridged species provided an exceptional profile, showing no significant change from the tested native antibody. Conjugates with significant disruption of the covalent connectivity of IgG1 chains resulted in a suboptimal Fc profile (CD16a kinetics or ADCC activity), in addition to sub-standard non Fc-specific attributes (thermal stability). These results advocate native disulfide re-bridging as an excellent synthetic strategy for forming homogenous IgG1 bioconjugates, with no reported negative impact on biophysical profile relative to the native antibody.
Linkers
that enable the site-selective synthesis of chemically
modified proteins are of great interest to the field of chemical biology.
Homogenous bioconjugates often show advantageous pharmacokinetic profiles
and consequently increased efficacy
in vivo
. Cysteine
residues have been exploited as a route to site-selectively modify
proteins, and many successfully approved therapeutics make use of
cysteine directed conjugation reagents. However, commonly used linkers,
including maleimide–thiol conjugates, are not stable to the
low concentrations of thiol present in blood. Furthermore, only a
few cysteine-targeting reagents enable the site-selective attachment
of multiple functionalities: a useful tool in the fields of theranostics
and therapeutic blood half-life extension. Herein, we demonstrate
the application of the pyridazinedione motif to enable site-selective
attachment of three functionalities to a protein bearing a single
cysteine residue. Extending upon previously documented dual modification
work, here we demonstrate that by exploiting a bromide leaving group
as an additional reactive point on the pyridazinedione scaffold, a
thiol or aniline derivative can be added to a protein, post-conjugation.
Thiol cleavability appraisal of the resultant C–S and C–N
linked thio-bioconjugates demonstrated C–S functionalized linkers
to be cleavable and C–N functionalized linkers to be noncleavable
when incubated in an excess of glutathione. The plug-and-play trifunctional
platform was exemplified by attaching clinically relevant motifs:
biotin, fluorescein, a polyethylene glycol chain, and a model peptide.
This platform provides a rare opportunity to combine up to three functionalities
on a protein in a site-selective fashion. Furthermore, by selecting
the use of a thiol or an amine for functionalization, we provide unique
control over linker cleavability toward thiols, allowing this novel
linker to be applied in a range of physiological environments.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.