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

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>

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 IC₅₀ 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

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