19 research outputs found
Attachment Site Cysteine Thiol p<i>K</i><sub>a</sub> Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody鈥揇rug Conjugates
The incorporation
of cysteines into antibodies by mutagenesis allows
for the direct conjugation of small molecules to specific sites on
the antibody via disulfide bonds. The stability of the disulfide bond
linkage between the small molecule and the antibody is highly dependent
on the location of the engineered cysteine in either the heavy chain
(HC) or the light chain (LC) of the antibody. Here, we explore the
basis for this site-dependent stability. We evaluated the in vivo
efficacy and pharmacokinetics of five different cysteine mutants of
trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide
bonds. A significant correlation was observed between disulfide stability
and efficacy for the conjugates. We hypothesized that the observed
site-dependent stability of the disulfide-linked conjugates could
be due to differences in the attachment site cysteine thiol p<i>K</i><sub>a</sub>. We measured the cysteine thiol p<i>K</i><sub>a</sub> using isothermal titration calorimetry (ITC) and found
that the variants with the highest thiol p<i>K</i><sub>a</sub> (LC K149C and HC A140C) were found to yield the conjugates with
the greatest in vivo stability. Guided by homology modeling, we identified
several mutations adjacent to LC K149C that reduced the cysteine thiol
p<i>K</i><sub>a</sub> and, thus, decreased the in vivo stability
of the disulfide-linked PBD conjugated to LC K149C. We also present
results suggesting that the high thiol p<i>K</i><sub>a</sub> of LC K149C is responsible for the sustained circulation stability
of LC K149C TDCs utilizing a maleimide-based linker. Taken together,
our results provide evidence that the site-dependent stability of
cys-engineered antibody-drug conjugates may be explained by interactions
between the engineered cysteine and the local protein environment
that serves to modulate the side-chain thiol p<i>K</i><sub>a</sub>. The influence of cysteine thiol p<i>K</i><sub>a</sub> on stability and efficacy offers a new parameter for the
optimization of ADCs that utilize cysteine engineering