12 research outputs found
FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody–Drug Conjugates
Despite the recent success of antibody–drug
conjugates (ADCs)
in cancer therapy, a detailed understanding of their entry, trafficking,
and metabolism in cancer cells is limited. To gain further insight
into the activation mechanism of ADCs, we incorporated fluorescence
resonance energy transfer (FRET) reporter groups into the linker connecting
the antibody to the drug and studied various aspects of intracellular
ADC processing mechanisms. When comparing the trafficking of the antibody–FRET
drug conjugates in various different model cells, we found that the
cellular background plays an important role in how the antigen-mediated
antibody is processed. Certain tumor cells showed limited cytosolic
transport of the payload despite efficient linker cleavage. Our FRET
assay provides a facile and robust assessment of intracellular ADC
activation that may have significant implications for the future development
of ADCs
FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody–Drug Conjugates
Despite the recent success of antibody–drug
conjugates (ADCs)
in cancer therapy, a detailed understanding of their entry, trafficking,
and metabolism in cancer cells is limited. To gain further insight
into the activation mechanism of ADCs, we incorporated fluorescence
resonance energy transfer (FRET) reporter groups into the linker connecting
the antibody to the drug and studied various aspects of intracellular
ADC processing mechanisms. When comparing the trafficking of the antibody–FRET
drug conjugates in various different model cells, we found that the
cellular background plays an important role in how the antigen-mediated
antibody is processed. Certain tumor cells showed limited cytosolic
transport of the payload despite efficient linker cleavage. Our FRET
assay provides a facile and robust assessment of intracellular ADC
activation that may have significant implications for the future development
of ADCs
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
Immolation of <i>p</i>‑Aminobenzyl Ether Linker and Payload Potency and Stability Determine the Cell-Killing Activity of Antibody–Drug Conjugates with Phenol-Containing Payloads
The valine-citrulline (Val-Cit) dipeptide
and <i>p</i>-aminobenzyl (PAB) spacer have been commonly
used as a cleavable
self-immolating linker in ADC design including in the clinically approved
ADC, brentuximab vedotin (Adcetris). When the same linker was used
to connect to the phenol of the cyclopropabenzindolone (CBI) (<b>P1</b>), the resulting <b>ADC1</b> showed loss of potency
in CD22 target-expressing cancer cell lines (e.g., BJAB, WSU-DLCL2).
In comparison, the conjugate (<b>ADC2</b>) of a cyclopropapyrroloindolone
(CPI) (<b>P2</b>) was potent despite the two corresponding free
drugs having similar picomolar cell-killing activity. Although the
corresponding spirocyclization products of <b>P1</b> and <b>P2</b>, responsible for DNA alkylation, are a prominent component
in buffer, the linker immolation was slow when the PAB was connected
as an ether (PABE) to the phenol in <b>P1</b> compared to that
in <b>P2</b>. Additional immolation studies with two other PABE-linked
substituted phenol compounds showed that electron-withdrawing groups
accelerated the immolation to release an acidic phenol-containing
payload (to delocalize the negative charge on the anticipated anionic
phenol oxygen during immolation). In contrast, efficient immolation
of <b>LD4</b> did not result in an active <b>ADC4</b> because
the payload (<b>P4</b>) had a low potency to kill cells. In
addition, nonimmolation of <b>LD5</b> did not affect the cell-killing
potency of its <b>ADC5</b> since immolation is not required
for DNA alkylation by the center-linked pyrrolobenzodiazepine. Therefore,
careful evaluation needs to be conducted when the Val-Cit-PAB linker
is used to connect antibodies to a phenol-containing drug as the linker
immolation, as well as payload potency and stability, affects the
cell-killing activity of an ADC
Linker Immolation Determines Cell Killing Activity of Disulfide-Linked Pyrrolobenzodiazepine Antibody–Drug Conjugates
Disulfide bonds could
be valuable linkers for a variety of therapeutic
applications requiring tunable cleavage between two parts of a molecule
(e.g., antibody–drug conjugates). The in vitro linker immolation
of β-mercaptoethyl-carbamate disulfides and DNA alkylation properties
of associated payloads were investigated to understand the determinant
of cell killing potency of anti-CD22 linked pyrrolobenzodiazepine
(PBD-dimer) conjugates. Efficient immolation and release of a PBD-dimer
with strong DNA alkylation properties were observed following disulfide
cleavage of methyl- and cyclobutyl-substituted disulfide linkers.
However, the analogous cyclopropyl-containing linker did not immolate,
and the associated thiol-containing product was a poor DNA alkylator.
As predicted from these in vitro assessments, the related anti-CD22
ADCs showed different target-dependent cell killing activities in
WSU-DLCL2 and BJAB cell lines. These results demonstrate how the in
vitro immolation models can be used to help design efficacious ADCs
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
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