9 research outputs found
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
Pyrrolobenzodiazepine Dimer Antibody–Drug Conjugates: Synthesis and Evaluation of Noncleavable Drug-Linkers
Three rationally designed pyrrolobenzodiazepine
(PBD) drug-linkers
have been synthesized via intermediate <b>19</b> for use in
antibody–drug conjugates (ADCs). They lack a cleavable trigger
in the linker and consist of a maleimide for cysteine antibody conjugation,
a hydrophilic spacer, and either an alkyne (<b>6</b>), triazole
(<b>7</b>), or piperazine (<b>8</b>) link to the PBD.
In vitro IC<sub>50</sub> values
were 11–48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (<b>7</b> inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and
0.10–1.73 μg/mL (<b>7</b> inactive) in CD22 3+
BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive
at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in
Founder 5 was observed with tumor stasis at 0.5–1 mg/kg, 1
mg/kg, and 3–6 mg/kg for <b>6</b>, <b>8</b>, and <b>7</b>, respectively. Tumor stasis at 2 mg/kg was observed for
anti-CD22 <b>6</b> in WSU-DLCL2. In summary, noncleavable PBD-ADCs
exhibit potent activity, particularly in HER2 models
An Anti-B7-H4 Antibody–Drug Conjugate for the Treatment of Breast Cancer
B7-H4
has been implicated in cancers of the female reproductive system and
investigated for its possible use as a biomarker for cancer, but there
are no preclinical studies to demonstrate that B7-H4 is a molecular
target for therapeutic intervention of cancer. We provide evidence
that the prevalence and expression levels of B7-H4 are high in different
subtypes of breast cancer and that only a few normal tissues express
B7-H4 on the cell membrane. These profiles of low normal expression
and upregulation in cancer provide an opportunity for the use of antibody–drug
conjugates (ADCs), cytotoxic drugs chemically linked to antibodies,
for the treatment of B7-H4 positive cancers. We have developed an
ADC specific to B7-H4 that uses a linker drug consisting of a potent
antimitotic, monomethyl auristatin E (MMAE), linked to engineered
cysteines (THIOMAB) via a protease labile linker. We will refer to
ADCs that use the THIOMAB format as TDCs to help distinguish the
format from standard MC-vc-MMAE ADCs that are conjugated to the interchain
disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced
durable tumor regression in cell line and patient-derived xenograft
models of triple-negative breast cancer. It also binds rat B7-H4 with
similar affinity to human and allowed us to test for target dependent
toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings
similar to untargeted TDC. Our results validate B7-H4 as an ADC target
for breast cancer and support the possible use of this TDC in the
treatment of B7-H4<sup>+</sup> breast cancer
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