4 research outputs found
Custom-Designed Affinity Capture LC-MS F(ab′)2 Assay for Biotransformation Assessment of Site-Specific Antibody Drug Conjugates
Affinity capture
liquid chromatography–mass spectrometry
(LC-MS) intact antibody assay has been widely used for direct drug-to-antibody
ratio (DAR) and catabolite characterization of antibody-drug conjugates
(ADCs). However, the intact mass spectra of new ADCs, which incorporate
new types of linkers and payloads other than maytansines and auristatins,
are more complex than those examined previously. The current method
has showed some limitations in elucidating certain structural modifications.
Herein, we report an alternative analytical approach for ADCs, such
as THIOMAB antibody-drug conjugates (TDCs), where the linker drugs
are site-specifically conjugated in the Fab region. The newly developed
affinity capture LC-MS FÂ(ab′)Â2 assay incorporates affinity
capture of human IgGs via binding to the Fab region, followed by on-bead
IdeS digestion to remove the Fc domain specifically and uniformly.
The resulting FÂ(ab′)Â2 (∼100 kDa) fragments contain the
key ADC biotransformation information, such as drug-to-antibody ratio
and drug metabolism and are more readily analyzed by electrospray
ionization LC-MS than the intact ADC (∼150 kDa). The reduced
size of analytes results in improved mass spectral sensitivity and
resolution. In addition, the reduced and optimized sample preparation
time, for example, rapid removal of the Fc fragment by IdeS digestion,
minimizes assay artifacts of drug metabolism and skewed DAR profiles
that may result from the prolonged incubation times (e.g., overnight
enzymatic treatment for Fc deglycosylation). The affinity capture
LC-MS FÂ(ab′)Â2 assay provides more detailed and accurate information
on ADC biotransformations in vivo, enabling analysis of low-dose,
labile, and complex site-specific ADCs with linker-drug conjugated
in the Fab region
Supplementation of Nicotinic Acid with NAMPT Inhibitors Results in Loss of In Vivo Efficacy in NAPRT1-Deficient Tumor Models
Nicotinamide adenine dinucleotide (NAD) is a metabolite essential for cell survival and generated de novo from tryptophan or recycled from nicotinamide (NAM) through the nicotinamide phosphoribosyltransferase (NAMPT)-dependent salvage pathway. Alternatively, nicotinic acid (NA) is metabolized to NAD through the nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1)-dependent salvage pathway. Tumor cells are more reliant on the NAMPT salvage pathway making this enzyme an attractive therapeutic target. Moreover, the therapeutic index of NAMPT inhibitors may be increased by in NAPRT-deficient tumors by NA supplementation as normal tissues may regenerate NAD through NAPRT1. To confirm the latter, we tested novel NAMPT inhibitors, GNE-617 and GNE-618, in cell culture- and patient-derived tumor models. While NA did not protect NAPRT1-deficient tumor cell lines from NAMPT inhibition in vitro, it rescued efficacy of GNE-617 and GNE-618 in cell culture- and patient-derived tumor xenografts in vivo. NA co-treatment increased NAD and NAM levels in NAPRT1-deficient tumors to levels that sustained growth in vivo. Furthermore, NAM co-administration with GNE-617 led to increased tumor NAD levels and rescued in vivo efficacy as well. Importantly, tumor xenografts remained NAPRT1-deficient in the presence of NA, indicating that the NAPRT1-dependent pathway is not reactivated. Protection of NAPRT1-deficient tumors in vivo may be due to increased circulating levels of metabolites generated by mouse liver, in response to NA or through competitive reactivation of NAMPT by NAM. Our results have important implications for the development of NAMPT inhibitors when considering NA co-treatment as a rescue strategy
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