Overcoming resistance remains a central challenge for antibody–drug conjugates (ADCs). Dual-payload strategies—incorporating two mechanistically distinct cytotoxic agents into a single ADC—are being actively explored to address tumor heterogeneity and adaptive repair mechanisms. At the same time, ADC plus small-molecule combination approaches are advancing in parallel, particularly with DNA damage response (DDR) inhibitors that sensitize tumors to DNA-targeting payloads. Both strategies share a common goal: to broaden efficacy and deliver more durable responses.

Colorectal cancer (CRC) offers a representative model to study these concepts because of its clinical prevalence and molecular diversity. Sensitivity to topoisomerase I (Topo1) inhibitors varies substantially across CRC models, reflecting one of the key challenges for payload-based therapies. To explore potential solutions, we selected a panel of nine agents spanning complementary mechanistic classes:

Topo1 inhibitors (DXd, Exatecan, SN-38): clinically validated payloads with differential activity across cancer models.

Tubulin inhibitor (MMAE): a microtubule-targeting payload with broad potency.

DDR pathway inhibitors: small molecules that disrupt DNA repair and checkpoint signaling, including WEE1 inhibitor (Azenosertib), CHK1/2 inhibitor (Prexasertib), PARP inhibitor (Talazoparib), ATR inhibitor (Ceralasertib), and ATM inhibitor (Lartesertib).

By systematically evaluating these compounds as single agents and in combination across a CRC cell panel, we aimed to highlight interactions with synergistic potential. Such data provide guidance not only for dual-payload ADC design but also for rational ADC + DDRi combination strategies, offering a broader translational framework for overcoming resistance in oncology.

CRC cell lines were cultured under recommended conditions and seeded into 384-well plates (40 µL/well). Compounds were prepared in DMSO as 3-fold serial dilutions (10 concentrations, 1000× stock) and dispensed at 40 nL per well (final DMSO 0.1%). Plates were incubated at 37 °C, 5% CO₂ for 3 days for DXd, Exatecan, SN-38, Azenosertib, Prexasertib, Lartesertib, Ceralasertib, and MMAE, or 7 days for Talazoparib single-agent assays (3 days for combinations). Cell viability was assessed using the Cell Counting-Lite 2.0 luminescent assay, with luminescence signals measured on a BMG plate reader to quantify ATP levels.

To establish a basis for dual-payload evaluation, we first profiled the single-agent activity of 9 representative compounds across 36 CRC cell lines. This step was designed to capture the intrinsic variability of drug responses in CRC and to identify models with reduced sensitivity to Topo1 inhibitors, which could serve as a context for testing combination effects.

The results confirmed heterogeneous responses. Based on these results, LS411N and T84 were identified as relatively insensitive to Topo1 inhibition and selected for subsequent dual-payload screening.

To investigate the potential of dual payload strategies in colorectal cancer, we established a combination screening assay using two representative CRC cell lines. In each model, Topo1 inhibitors (DXd or Exatecan) were selected as anchor payloads and systematically combined with a panel of additional agents. This design enabled us to rapidly identify combinations with enhanced activity profiles and highlight a subset of promising pairs.

Complete data available upon request.

These findings provided the rationale to advance selected combinations into dedicated synergy matrix assays for quantitative assessment.

Based on the preliminary IC50 screening, we selected six dual-payload pairs across two colorectal cancer cell lines (T84 and LS411N) for further validation. These combinations include Topo1 inhibitors (DXd or Exatecan) paired with DDR modulators (Prexasertib, Ceralasertib, Azenosertib) under different fixed-dose conditions.

3X denotes 1:3 serial dilutions from the listed top concentration.

Combination matrix assays are used to quantitatively assess the interaction between payloads. Synergy will be assessed using the HSA model, enabling us to distinguish between additive effects and true synergy.

Synergy evaluation of dual payload combinations in LS411N cells.

Synergy evaluation of dual payload combimnations in T84 cells.

Topo1 inhibitors induce DNA double-strand breaks during replication, but cancer cells frequently repair this damage through DDR pathways. Blocking these checkpoints disables repair capacity, forcing tumor cells into catastrophic replication stress and cell death. This provides a strong mechanistic basis for combining Topo1 inhibitors with DDR modulators—whether within dual-payload ADC constructs or as ADC plus small-molecule DDRi combinations.

Our systematic screening in colorectal cancer cell lines revealed robust synergy between Topo1 inhibitors and DDR modulators, with four combinations in two cell lines consistently achieving HSA synergy scores >10. These findings align with published CRC data: Exatecan and Ceralasertib synergize in HCT116 colon cancer models and xenografts; ATR inhibition enhances irinotecan efficacy in CRC cells and xenografts in a schedule-dependent manner; SN-38 shows strong synergy with CHK1 inhibition across CRC models; and Prexasertib selectively targets CRC stem-like cells with replication stress and DNA repair deficiencies. Clinically, early-phase studies are also evaluating trastuzumab deruxtecan (T-DXd) combined with Ceralasertib, including CRC cohorts.

Together, these findings provide a strong mechanistic and translational rationale for dual-payload strategies that incorporate Topo1 inhibitors with complementary mechanisms, as well as for ADC + DDRi combination strategies already advancing in the clinic.

Importantly, the synergy observed with DXd and Prexasertib has not been previously reported, highlighting a novel opportunity for further investigation.

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