3 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–Drug 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
Modulating Antibody–Drug Conjugate Payload Metabolism by Conjugation Site and Linker Modification
Previous investigations
on antibody-drug conjugate (ADC) stability
have focused on drug release by linker-deconjugation due to the relatively stable payloads such
as maytansines. Recent development of ADCs has been focused on exploring
technologies to produce homogeneous ADCs and new classes of payloads
to expand the mechanisms of action of the delivered drugs. Certain
new ADC payloads could undergo metabolism in circulation while attached
to antibodies and thus affect ADC stability, pharmacokinetics, and
efficacy and toxicity profiles. Herein, we investigate payload stability
specifically and seek general guidelines to address payload metabolism
and therefore increase the overall ADC stability. Investigation was
performed on various payloads with different functionalities (e.g.,
PNU-159682 analog, tubulysin, cryptophycin, and taxoid) using different
conjugation sites (HC-A118C, LC-K149C, and HC-A140C) on THIOMAB antibodies.
We were able to reduce metabolism and inactivation of a broad range
of payloads of THIOMAB antibody-drug conjugates by employing optimal
conjugation sites (LC-K149C and HC-A140C). Additionally, further payload
stability was achieved by optimizing the linkers. Coupling relatively
stable sites with optimized linkers provided optimal stability and
reduction of payloads metabolism in circulation in vivo
Exploration of Pyrrolobenzodiazepine (PBD)-Dimers Containing Disulfide-Based Prodrugs as Payloads for Antibody–Drug Conjugates
A number
of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing
various disulfide-based prodrugs were evaluated for their ability
to undergo activation (disulfide cleavage) <i>in vitro</i> in the presence of either glutathione (GSH) or cysteine (Cys). A
good correlation was observed between <i>in vitro</i> GSH
stability and <i>in vitro</i> cytotoxicity toward tumor
cell lines. The prodrug-containing compounds were typically more potent
against cells with relatively high intracellular GSH levels (e.g.,
KPL-4 cells). Several antibody–drug conjugates (ADCs) were
subsequently constructed from PBD dimers that incorporated selected
disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation
activity against KPL-4 cells <i>in vitro</i> in an antigen-dependent
manner. However, the disulfide prodrugs contained in the majority
of such entities were surprisingly unstable toward whole blood from
various species. One HER2-targeting conjugate that contained a thiophenol-derived
disulfide prodrug was an exception to this stability trend. It exhibited
potent activity in a KPL-4 <i>in vivo</i> efficacy model
that was approximately three-fold weaker than that displayed by the
corresponding parent ADC. The same prodrug-containing conjugate demonstrated
a three-fold improvement in mouse tolerability properties <i>in vivo</i> relative to the parent ADC, which did not contain
the prodrug