5 research outputs found
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
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
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