9 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 F(ab′)2 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 F(ab′)2 (∼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 F(ab′)2 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
High-Resolution Accurate-Mass Mass Spectrometry Enabling In-Depth Characterization of <i>in Vivo</i> Biotransformations for Intact Antibody-Drug Conjugates
Antibody-drug
conjugates (ADCs) represent a promising class of
therapeutics for the targeted delivery of highly potent cytotoxic
drugs to tumor cells to improve bioactivity while minimizing side
effects. ADCs are composed of both small and large molecules and therefore
have complex molecular structures. <i>In vivo</i> biotransformations
may further increase the complexity of ADCs, representing a unique
challenge for bioanalytical assays. Quadrupole-time-of-flight mass
spectrometry (Q-TOF MS) with electrospray ionization has been widely
used for characterization of intact ADCs. However, interpretation
of ADC biotransformations with small mass changes, for the intact
molecule, remains a limitation due to the insufficient mass resolution
and accuracy of Q-TOF MS. Here, we have investigated <i>in vivo</i> biotransformations of multiple site-specific THIOMAB antibody-drug
conjugates (TDCs), in the intact form, using a high-resolution, accurate-mass
(HR/AM) MS approach. Compared with conventional Q-TOF MS, HR/AM Orbitrap
MS enabled more comprehensive identification of ADC biotransformations.
It was particularly beneficial for characterizing ADC modifications
with small mass changes such as partial drug loss and hydrolysis.
This strategy has significantly enhanced our capability to elucidate
ADC biotransformations and help understand ADC efficacy and safety <i>in vivo</i>
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
Development of Efficient Chemistry to Generate Site-Specific Disulfide-Linked Protein– and Peptide–Payload Conjugates: Application to THIOMAB Antibody–Drug Conjugates
Conjugation
of small molecule payloads to cysteine residues on
proteins via a disulfide bond represents an attractive strategy to
generate redox-sensitive bioconjugates, which have value as potential
diagnostic reagents or therapeutics. Advancement of such “direct-disulfide”
bioconjugates to the clinic necessitates chemical methods to form
disulfide connections efficiently, without byproducts. The disulfide
connection must also be resistant to premature cleavage by thiols
prior to arrival at the targeted tissue. We show here that commonly
employed methods to generate direct disulfide-linked bioconjugates
are inadequate for addressing these challenges. We describe our efforts
to optimize direct-disulfide conjugation chemistry, focusing on the
generation of conjugates between cytotoxic payloads and cysteine-engineered
antibodies (i.e., THIOMAB antibody–drug conjugates, or TDCs).
This work culminates in the development of novel, high-yielding conjugation
chemistry for creating direct payload disulfide connections to any
of several Cys mutation sites in THIOMAB antibodies or to Cys sites
in other biomolecules (e.g., human serum albumin and cell-penetrating
peptides). We conclude by demonstrating that hindered direct disulfide
TDCs with two methyl groups adjacent to the disulfide, which have
heretofore not been described for any bioconjugate, are more stable
and more efficacious in mouse tumor xenograft studies than less hindered
analogs
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
Discovery of Peptidomimetic Antibody–Drug Conjugate Linkers with Enhanced Protease Specificity
Antibody–drug
conjugates (ADCs) have become an important
therapeutic modality for oncology, with three approved by the FDA
and over 60 others in clinical trials. Despite the progress, improvements
in ADC therapeutic index are desired. Peptide-based ADC linkers that
are cleaved by lysosomal proteases have shown sufficient stability
in serum and effective payload-release in targeted cells. If the linker
can be preferentially hydrolyzed by tumor-specific proteases, safety
margin may improve. However, the use of peptide-based linkers limits
our ability to modulate protease specificity. Here we report the structure-guided
discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing
linker is hydrolyzed predominantly by cathepsin B while the valine–citrulline
dipeptide linker is not. ADCs bearing the nonpeptidic linker are as
efficacious and stable in vivo as those with the dipeptide linker.
Our results strongly support the application of the peptidomimetic
linker and present new opportunities for improving the selectivity
of ADCs
Discovery of Peptidomimetic Antibody–Drug Conjugate Linkers with Enhanced Protease Specificity
Antibody–drug
conjugates (ADCs) have become an important
therapeutic modality for oncology, with three approved by the FDA
and over 60 others in clinical trials. Despite the progress, improvements
in ADC therapeutic index are desired. Peptide-based ADC linkers that
are cleaved by lysosomal proteases have shown sufficient stability
in serum and effective payload-release in targeted cells. If the linker
can be preferentially hydrolyzed by tumor-specific proteases, safety
margin may improve. However, the use of peptide-based linkers limits
our ability to modulate protease specificity. Here we report the structure-guided
discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing
linker is hydrolyzed predominantly by cathepsin B while the valine–citrulline
dipeptide linker is not. ADCs bearing the nonpeptidic linker are as
efficacious and stable in vivo as those with the dipeptide linker.
Our results strongly support the application of the peptidomimetic
linker and present new opportunities for improving the selectivity
of ADCs
Discovery of Peptidomimetic Antibody–Drug Conjugate Linkers with Enhanced Protease Specificity
Antibody–drug
conjugates (ADCs) have become an important
therapeutic modality for oncology, with three approved by the FDA
and over 60 others in clinical trials. Despite the progress, improvements
in ADC therapeutic index are desired. Peptide-based ADC linkers that
are cleaved by lysosomal proteases have shown sufficient stability
in serum and effective payload-release in targeted cells. If the linker
can be preferentially hydrolyzed by tumor-specific proteases, safety
margin may improve. However, the use of peptide-based linkers limits
our ability to modulate protease specificity. Here we report the structure-guided
discovery of novel, nonpeptidic ADC linkers. We show that a cyclobutane-1,1-dicarboxamide-containing
linker is hydrolyzed predominantly by cathepsin B while the valine–citrulline
dipeptide linker is not. ADCs bearing the nonpeptidic linker are as
efficacious and stable in vivo as those with the dipeptide linker.
Our results strongly support the application of the peptidomimetic
linker and present new opportunities for improving the selectivity
of ADCs
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