Antibody–Exatecan Conjugates with a Novel Self-immolative Moiety Overcome Resistance in Colon and Lung Cancer

Antibody–drug conjugates (ADC) provide targeted tumor-cell delivery of cytotoxic drugs (“payloads”) on antibody carriers. With the approval of more than 10 drugs (7 within the last few years) and more in clinical development, ADCs are undergoing a renaissance (1). This breakthrough is largely driven by a transformative ADC design that uses less potent but safer payload DNA topoisomerase I (TOP1) inhibitor camptothecins (SN38 or DXd) and a higher drug load (DAR 8) to increase therapeutic index, allowing the administration of higher doses of ADC (∼5–10 mg/kg) to improve both drug delivery and tissue penetration without greater toxicity (2). For example, HER2-targeting T-DXd/DS-8201a (Enhertu; Daiichi Sankyo) is composed of highly stable peptide-linker GGFG and payload DXd, which is a derivative of camptothecin exatecan that is 10 to 100 times less potent than microtubule-targeting or DNA-alkylating agents. However, its strong bystander-killing effect overcomes resistance to T-DM1/trastuzumab (Tras), making it the first ADC effective against low HER2 expression tumors (3, 4). IMMU-132 (sacituzumab govitecan/Trodelvy; Gilead Sciences) targeting TROP2 using another TOP1 inhibitor SN-38 as the payload was the first effective ADC for triple-negative breast cancer (5). Additional GGFG–DXd ADCs, including U3-1402 (P–GGFG–DXd) targeting HER3 and datopotamab DXd (Dato-DXd) targeting TROP2, have shown promising therapeutic outcomes in patients, highlighting the unprecedented clinical success of using camptothecin analogues as ADC payload (6, 7).

Despite the breakthrough, DXd and SN-38 ADCs face intrinsic and acquired resistance due to tumor heterogeneity and multidrug resistance (MDR) regulation (8, 9). The typical treatment response rate among patients for payload-sensitive tumor types is only 30% to 50% due to tumor heterogeneity, as low target expression or high intratumoral heterogeneity results in more resistant tumors. DS-8201a generates an initial response of less than 50% and less than 30% in HER2-low-expressing breast and gastric cancers, respectively (10, 11). In non–small cell lung cancer (NSCLC), DS-8201a has improved the response rate of T-DM1 in HER2-overexpressing (IHC 3+ and 2+) patients, but objective response rate (ORR) is still less than 30% (12). DXd ADCs targeting HER3 and TROP2 are similarly less effective in tumors with low target expression in preclinical studies (6, 13). In clinical trials, U3-1402 is effective in patients with HER3-expressing, EGFR-mutated NSCLC, with an ORR of 39%, and patients with higher HER3 expression tend to have better responses (7). TROP2 downregulation due to target mutation after initial responses can cause an acquired resistance for Trodelvy (14). Furthermore, certain tumor types may be more challenging for DXd ADCs, as no meaningful response is observed for the majority of colon cancer cases (HER2 low) in the DS-8201a trial (15). In addition, DXd/SN-38 ADCs may be ineffective in MDR⁺ tumors due to a higher DXd/SN-38 efflux by the MDR transporter ABCG2 or P-gp (16, 17). Delineation of the inherent resistance mechanism associated with the current TOP1 inhibitor–based ADC class and the design of a limitation-overcoming ADC are clinically imperative.

Exatecan, a camptothecin and the precursor of DXd, has attracted initial attention due to its higher TOP1 inhibition potency (18, 19), lower sensitivity to ABCG2 or P-gp (20), and higher permeability and bystander penetration (21). However, exatecan is too hydrophobic to be conjugated directly to antibodies (22). Commonplace hydrophilic structures such as the β-glucuronidase linker or a peptide-linker modified with polyethylene glycol (PEG) are often used for masking exatecan (23–25). However, β-glucuronidase linkers have not advanced in clinical development, and the significant structural and chemistry changes in dipeptide linker Val-Ala (VA) or Val-Cit (VC) may affect their clinical developability (24–26). As linker chemistry design, payload conjugation strategy, and release mechanism affect physicochemical and pharmacologic performance of an ADC, a holistic approach for ADC design, mechanistic investigation, and a comprehensive comparison of an antibody–exatecan conjugate with the current ADC class are required to achieve successful clinical translation and authentic performance improvement.

To accomplish this goal, here we designed an antibody–exatecan conjugate class with an improved therapeutic index over DXd/SN-38 ADCs by biochemically and pharmacologically profiling exatecan versus DXd/SN-38 as an ADC payload. A novel chemistry (T moiety) compatible with clinically proven ADC linkers was introduced to mask exatecan hydrophobicity for traceless conjugation and exatecan release. T moiety (T800–T1000) translated a superior exatecan payload feature into ADCs, with improved physicochemical and pharmacologic performance demonstrating higher stability, deeper tumor penetration, and more durable antitumor responses than DXd/SN-38 ADCs. T moiety–exatecan ADCs were more effective against large-size tumors due to a higher intratumor pharmacodynamic response and were able to overcome MDR⁺ (high ABCG2/P-gp expression) tumor resistance to DXd/SN-38 ADCs in vivo, including challenging patient-derived xenograft (PDX) and organoid models. Despite higher potency, T moiety–exatecan ADCs did not incur higher toxicity in nonhuman primates. Initial exploration of combination therapies suggested that exatecan and its ADC had improved synergistic efficacy with DNA damage response (DDR) pathway inhibitors in colon cancer. Our data suggest that T moiety–exatecan ADCs may overcome resistance mechanisms to elicit responses from a larger patient population and a broader variety of tumors compared with prior ADCs.

DXd was derived from exatecan by the chemical modification of NH₂ on the F-ring by a 2-hydroxyacetyl (COCH₂OH) group (Supplementary Fig. S1A). Exatecan showed an average of 10 to 20 times more in vitro cytotoxic potency than DXd with subnanomolar IC₅₀ (Supplementary Fig. S1B; Supplementary Table S1) in a panel of cancer cell lines.

Cell-based TOP1 inhibition assays and structural modeling provided a mechanism of higher cytotoxic potency of exatecan. The level of DNA-trapped TOP1 (TOP1ccs) of exatecan in comparison with DXd and SN-38 was examined by a modified rapid approach to DNA adduct recovery (RADAR) assay (21). Exatecan was the most potent drug and induced TOP1ccs at a lower concentration than DXd and SN-38 (Fig. 1A; Supplementary Fig. S1C). DNA-trapped TOP1 is typically removed from DNA and degraded. To measure the rate of TOP1 degradation, cells were treated with exatecan or DXd/SN-38 for 2 hours and then allowed to grow without drugs for 30 minutes for the reversal of TOP1ccs and TOP1 degradation. Exatecan induced TOP1 degradation in a dose-dependent manner and was more effective than DXd and SN-38 (Fig. 1B).

Camptothecins trap TOP1ccs by π–π stacking with base pairs flanking the DNA cleavage site and by three hydrogen bonds with TOP1 residues (R364, D533, and N722; Fig. 1C; ref. 27). Docking simulation suggested that the amino group of exatecan may form two additional hydrogen bonds with the DNA base and the N352 residue of TOP1 (Fig. 1C). This interaction, however, was eliminated with the 2-hydroxyacetyl replacement of the amino group in DXd, possibly contributing to a decreased TOP1cc activity.

Lipophilic ligand efficiency (LLE) measures in vitro potency and lipophilicity. Increasing LLE values has been used to optimize absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics (28). Exatecan showed the highest LLE value of 10.08 compared with SN-38 (6.41) and DXd (7.61), reflecting improved “drug-like” properties of exatecan in terms of increased activity and lowered lipophilicity (Supplementary Fig. S1D).

The MDR transporter ABCG2- and P-gp–mediated efflux of exatecan, DXd, and SN-38 was determined in Caco-2 cells (Supplementary Fig. S1E). DXd showed an order of magnitude higher efflux ratios than exatecan without or with the ABCG2 inhibitor novobiocin, the P-gp inhibitor verapamil, or the dual inhibitor GF120918 (Fig. 1D). Interestingly, SN-38 efflux ratios were higher than exatecan but lower than DXd. Immunofluorescence (IF) examination of the intracellular accumulation of exatecan and DXd showed that DXd accumulation was lower than exatecan in the ABCG2 high expression cell line NCI-H460 (Fig. 1E; Supplementary Fig. S1F–1J).

Consistent with MDR substrate experiment results, the cytotoxic potency difference between DXd and exatecan showed a correlation with endogenous ABCG2/P-gp expression (mRNA and protein; Supplementary Fig. S2A–S2C). In general, the IC₅₀ ratio of DXd/exatecan was higher in cells with a higher ABCG2 or P-gp expression (Fig. 1F; Supplementary Fig. S2D and S2E). The inhibition of ABCG2 or P-gp improved the cytotoxicity of DXd/SN-38 but had a much smaller effect on exatecan (Fig. 1G). Even with an improved IC₅₀ with inhibitor for DXd/SN-38, exatecan remained more potent (Supplementary Fig. S2F and S2G). Collectively, exatecan demonstrated less sensitivity to the MDR genes than DXd/SN38, potentially conferring higher cytotoxicity in cancer cells along with a more prevalent resistance mechanism.

We conducted a 4-week (days 1, 8, 15, 22, and 29) intermittent intravenous dose toxicity study of exatecan mesylate in rats (6 animals/group) with a 4-week recovery period (3 of the 6 animals). Exatecan mesylate was well tolerated in rats at doses up to 10 mg/kg (calculated as exatecan-free base). At 30 mg/kg, exatecan mesylate resulted in decreased animal body weight, morbidity (decreased food consumption, abnormal clinical signs), and/or mortality (occurred 5 or 6 days after dose). The dose-dependent transient suppression of body weight gain (Supplementary Fig. S3A), decrease of blood cell counts (Supplementary Fig. S3B–S3E), and increase of key serum enzyme levels (Supplementary Fig. S3F and S3G) were observed in 3 mg/kg and 10 mg/kg groups but largely reversible by the end of the 4-week recovery period (Supplementary Table S2), suggesting that exatecan was similarly tolerated to DXd or SN-38 (16).

To enable a traceless conjugation and release of exatecan, we used a dipeptide VA linker and a modified self-immolative spacer p-amino benzyl (pAB) called T moiety (Fig. 2A; S1 in Supplementary Fig. S4A). VA was selected because it creates more hydrophilic high-DAR ADCs compared with other peptide linkers VC or Gly–Gly–Phe–Gly (GGFG; S0 in Supplementary Fig. S4A; refs. 29–30). Direct conjugation of HER2-targeting Tras and exatecan using the unmodified peptide linker VA/VC/GGFG caused high aggregation (Supplementary Fig. S4B). Integrating T moiety with a PEG group (T900; S3 in Supplementary Fig. S4A) significantly reduced aggregation to an acceptable level of 2%. However, modification of MC with the same PEG (called M moiety, M900; S2 in Supplementary Fig. S4A) led to a high aggregation of >50% (Supplementary Fig. S4B), highlighting the importance of the judicious selection of modification position. Considering the potential lethal adverse reactions caused by PEG-based biological drugs (31), we decided to use polysarcosine (pSAR) due to its higher solubility and biocompatibility (Supplementary Fig. S4C; ref. 32). Polysarcosine with 10 units (pSAR10) modified pAB (T1000) resulted in homogeneous ADC with a negligible level of aggregation (Supplementary Fig. S4B). Remarkably, an intermediate structure T800 with m-methylaminomethyl modification of pAB for attaching pSAR10 produced homogeneous ADC of high DAR without aggregation. In comparison, the attachment of the same m-methylaminomethyl group to the VA linker (VK-pABC, M800, CLogP = 4.98) caused 10% aggregation with exatecan even when T800 (CLogP = 4.73) and M800 are considered to be chemically equivalent, supporting pAB selection as the optimal modification site. The conjugation yield for T moiety ADCs was >90%. Replacing the VA linker with GGFG in T1000 also yielded homogeneous ADC without aggregation (T1001; Supplementary Fig. S4B).

To compare the physicochemical parameters of Tras–T1000–exatecan (MTX-1000) with those of DS-8201a, we used either purchased DS-8201a or internally manufactured Tras–GGFG–DXd with closely matched physicochemical profiles (Supplementary Fig. S5A and S5B), in vivo potency (Supplementary Fig. S5C–S5E), and pharmacokinetics (PK) in mice (Supplementary Fig. S5F). The attachment of exatecan to the HER2 antibody did not affect HER2 antibody binding to target by flow cytometry or ELISA, similar to Tras–GGFG–DXd (Supplementary Fig. S6A and S6B). Tras–exatecan conjugates using T800 or T900 also resulted in more hydrophilic ADCs than Tras–GGFG–DXd (Supplementary Fig. S6C and S6D). Both DAR8 and DAR4 ADCs were readily generated (Supplementary Fig. S6E).

Tras–T moiety–exatecan ADCs (Tras–T800–exatecan/MTX-800 and Tras–T1000–exatecan/MTX-1000, both DAR8) demonstrated similar or better stability than Tras–GGFG–DXd. This was measured by aggregation formed for an extended incubation time at 37°C and under repeat freeze–thaw cycles (Supplementary Fig. S6F). T moiety–exatecan ADCs also showed better thermostability and photostability than DS-8201a (Supplementary Fig. S6G and S6H). Notably, VA and pSAR ADC (T1000) showed better stability and less aggregation than other linker (GGFG) and modification (PEG; T900 or T1001; Supplementary Fig. S6C, S6D, S6F, and S6G), highlighting the impact of linker and conjugation chemistry choice on the physicochemical function of an ADC.

T moiety–exatecan-derived ADCs targeting HER3 with patritumab (P; DAR 8), TROP2 using datopotamab (Dato; Dato–T1000–exatecan) or hRS7 antibody from Trodelvy (MTX-132; DAR 4), and CDH6 using DS-6000 antibody (DS6000; see Methods) were more hydrophilic than DXd-based ADCs using the same antibodies, and all ADCs were homogeneous without aggregation [Supplementary Fig. S7A–S7C; hydrophobic interaction chromatography (HIC), size-exclusion chromatography]. T1000 was successfully used to build homogeneous, hydrophilic ADC using Tras and another TOP1 inhibitor belotecan (Supplementary Fig. S7D and S7E). HER2-targeting belotecan ADC showed potent, specific in vitro cytotoxicity in a HER2-high N87 cell line (no activity in HER2-negative MDA-MB-468; Supplementary Fig. S7F). Together, T moiety was proven to be useful for conjugating challenging cytotoxic compounds as ADC payloads.

The release rate of exatecan from MTX-1000 ranged from 0.4% to 3.6% on day 14 in human, monkey, rat, and mouse plasma by immunocapture followed by reverse-phase LC/MS-MS, compared with 1.4% to 6.6% for Tras–GGFG–DXd (Fig. 2B; Supplementary Fig. S8A), consistent with antibody and ADC stability data (Supplementary Fig. S8B and S8C; Supplementary Table S3). MTX-1000 in particular released more than 10 times less payload in human/monkey plasma than Tras–GGFG–DXd (Fig. 2B; Supplementary Table S3). In vivo, after a single intravenous administration of MTX-1000 at 4 mg/kg in rats, the PK profile of MTX-1000 was similar to that of total antibody (Fig. 2C). MTX-1000 is more stable than Tras–GGFG–DXd, as demonstrated by its longer half-life (T_1/2 4.14 ± 1.45 for Tras–GGFG–DXd vs. 7.4 ± 1.5 for MTX-1000) and lower payload release, which resulted in over 50% more AUC for both total antibody and ADC (Supplementary Fig. S8D and S8E; Supplementary Table S4). The higher stability of MTX-1000 was consistent with the more hydrophilic nature of the molecule.

The internalization rate of MTX-1000 in cancer cell lines was similar to Tras–GGFG–DXd (Supplementary Fig. S9A–S9C; Supplementary Table S5). There was an inverse relationship between HER2 expression level and internalization rate, consistent with a previous report (33). Twenty-four hours after MTX-1000 was internalized into the cells, the quantity of exatecan released in the culture media correlated with the cell-surface HER2 expression level of each cell line (Fig. 2D; Supplementary Table S6). However, DS-8201a showed a higher DXd release in the culture media regardless of HER2 expression due to a high background DXd release unrelated to cell binding and internalization, as shown by a 20- to 40-fold payload release increase in the media (without cells) compared with MTX-1000 at 8 and 24 hours, respectively (Supplementary Fig. S9D and S9E).

MTX-1000 demonstrated a superior pharmacologic profile characterized by higher potency and deeper penetration. MTX-1000 induced higher cytotoxic TOP1ccs and faster TOP1 degradation compared with DS-8201a (Fig. 2E and F; Supplementary Fig. S9F and S9G). The TOP1 inhibition effect of MTX-1000 was confirmed by DNA damage (phosphorylations of Chk1 and histone γH2A.X) and apoptosis (cleaved PARP) in HCC1954 cells in the presence of MTX-1000, exatecan (positive), and Tras (negative; Supplementary Fig. S9H). To gain cellular resolution of ADC potency and payload distribution, tumor penetration of MTX-1000 and DS-8201a was evaluated by cell imaging and HER2-positive patient-derived colon cancer organoids (PDO; refs. 21, 34; Supplementary Fig. S10A and S10B). In a PDO, MTX-1000 caused more cell death to prevent organoid growth than DS-8201a (Fig. 2G and H; Supplementary Fig. S10C–S10E). Notably, the cell death signals (PI staining) of MTX-1000 extended from surface to the core of the organoids compared with mostly peripheral staining for DS-8201a (Fig. 2I; Supplementary Fig. S10F and S10G). Similar results were observed in another colon cancer organoid (Supplementary Fig. S10H).

MTX-1000 showed a higher bystander killing efficacy than Tras–GGFG–DXd in an in vitro coculture system of HER2-negative (MDA-MB-468) and HER2-positive (HCC1954) cell lines (Fig. 2J; Supplementary Fig. S11A–S11D), supported by a 10-fold increase in the cell permeability (A–B) of exatecan compared with DXd/SN-38 (Supplementary Fig. S1E).

A CellTiter-Glo 3D assay was performed to evaluate the potential myelotoxicity of ADC and payload. The IC₅₀ of MTX-1000, DS-8201, and Trodelvy was 1.24 μg/mL, 1.47 μg/mL, and 0.15 μg/mL, respectively (Fig. 2K, left). MTX-1000 and DS-8201a showed comparable myelotoxicity despite the 5-fold myelotoxicity potential difference between exatecan and DXd (Fig. 2K, middle and right). MTX-1000's higher stability likely allowed for its decreased myelotoxicity. Trodelvy showed much lower IC₅₀ due to known instability in releasing payload SN-38.

MTX-1000 toxicity was evaluated in an exploratory cynomolgus monkey (cross-reactive species) toxicity study (every 3 weeks × 3 at 10 mg/kg and 30 mg/kg). The target organ of toxicity is summarized in Table 1. Primary toxicity findings were found only in lymphatic/hematopoietic organs and digestive systems in monkeys. All findings were minimal or mild, and almost all treatment-related changes showed recovery or a trend of reversibility after a 1-week withdrawal period. Reticulocyte counts showed decreases and rebound beyond the normal range, whereas other hematologic indicator changes were largely within the normal range (Supplementary Fig. S12A–S12G). Serum chemistry also remained normal (Supplementary Fig. S12H and S12I). The manifested digestive system toxicity symptom was limited to only mild diarrhea. Gastrointestinal (GI) toxicity and bone marrow toxicity are typical dose-limiting factors in the clinical use of TOP1 inhibitors, but the effects of MTX-1000 on the intestines were minimal. Based on the toxicology findings, the highest nonseverely toxic dose (HNSTD) for monkeys was considered to be 30 mg/kg, the same as DS-8201a. However, macroscopic toxicity findings in the intestines, bone marrows, and particularly in the lung shown in DS-8201a studies were not observed (Supplementary Fig. S12J). These findings suggest safety profiles of MTX-1000 are similar to or better than those of Tras–GGFG–DXd despite higher antitumor potency. Similarly, TROP2-targeting MTX-132 also displayed mild clinical signs only (skin discolored at forelimb and hindlimb) and its HNSTD was judged to be >30 mg/kg compared with a reported HNSTD of 10 mg/kg for Dato–DXd (7).

In vitro, HER-targeting MTX-1000, TROP2-targeting MTX-132, and HER3-targeting P–T1000–exatecan were more cytotoxic than their respective counterpart DXd ADC, especially in low-target-expression cells (Supplementary Fig. S13A–S13C; Supplementary Table S7).

Monkey HNSTD or the mouse PK method was used to assess the therapeutic index of MTX-1000 and MTX-132 against DS-8201a and Trodelvy/Dato–DXd (35). First, minimum effective doses (MED) for inducing tumor regression in vivo were measured by dose–efficacy titration. In HCC1954 with a medium HER2 expression, the MED of MTX-1000 was about one third of DS-8201a (Supplementary Fig. S13D). However, in the HER2-low-expression model COLO205, only 10 mg/kg of MTX-1000 caused tumor regression, whereas 10 mg/kg of DS-8201a was only partially effective (Supplementary Fig. S13E). In the TROP2-high-expression model H2170, the MED of MTX-132 was (single dose) less than 5 mg/kg compared with four doses of 25 mg/kg of Trodelvy (Supplementary Fig. S13F). Dato–GGFG–DXd was less effective even at 10 mg/kg (Supplementary Fig. S13F). In COLO205 with a medium TROP2 expression, 10 mg/kg of MTX-132 produced a tumor regression, but four doses of 25 mg/kg of Trodelvy did not (Supplementary Fig. S13G). Combining HNSTD data from monkey studies, we found that MTX-1000 and MTX-132 improved the therapeutic index two to five times compared with DS-8201a and Trodelvy/Dato–DXd in medium- to high-target-expression models (Supplementary Fig. S13H). The therapeutic index measured by the mouse PK (AUC) of DS-8201a and MTX-1000 yielded a 2-fold improvement (Supplementary Fig. S13H and S13I). In low target expression, tumors resistant to DS-8201a or Trodelvy treatment, MTX-1000, and MTX-132 showed a more significant therapeutic index advantage (Fig. 3A).

Supported by an improved therapeutic index, T moiety–exatecan ADCs showed superior in vivo antitumor efficacy and a more durable response in clinically relevant PDX models. In a gastric PDX model with high HER expression, MTX-1000 inhibited tumor growth better than Tras–GGFG–DXd, although both ADCs produced only modest inhibition (Supplementary Fig. S14A). In the same gastric PDX model, MTX-132 (DAR 4) showed significantly better tumor inhibition than Dato–GGFG–DXd (DAR 4), with a tumor growth inhibition (TGI) of 65% versus 24% (Supplementary Fig. S14B). In a pancreatic PDX model with moderate HER2 expression, MTX-1000 and MTX-800 initially showed comparable tumor inhibition as Tras–GGFG–DXd but produced more durable tumor inhibition than Tras–GGFG–DXd over 50 days (Supplementary Fig. S14C and S14D). It was noteworthy that in two breast cancer PDX models, Tras–GGFG–DXd and Dato–DXd produced similar levels of tumor inhibition as MTX-1000 and MTX-132 (Supplementary Fig. S14E and S14F), reflecting the clinic observation that breast cancer has been one of the most responsive tumor types for DS-8201a and TROP2-targeting DXd/SN-38 ADCs.

In low-target-expression tumors, T1000–exatecan ADCs overcame tumor-intrinsic resistance to DXd/SN-38 ADCs. In a CDH6-low-expression (IHC 1+) PDX model of nephroblastoma with hepatic metastasis resistant to DS-6000, T moiety–exatecan ADC using DS-6000 antibody (DS6000–T1000–exatecan, DAR 8) showed strong antitumor activity (Fig. 3B). In H2170 with low HER3 expression, P–GGFG–DXd was ineffective (similar to PBS control) whereas P–T1000–exatecan produced tumor regression (Fig. 3C). COLO205 tumors outgrowth after DS-8201a treatment was tested to see whether T1000–exatecan ADCs could overcome resistance in DXd/SN-38 ADC–treated tumors (Fig. 3D). Those tumors were allowed to grow to 900 mm³ after two doses of DS-8201a. Subsequent administration of one dose of MTX-1000 produced significant tumor regression, whereas one dose of DS-8201a was ineffective (Fig. 3D). MTX-1000 was also effective in growing tumors (at 600 mm³) after DS-8201a treatment in another colon cancer xenograft model, HCT-15 (Supplementary Fig. S15A). Similarly, MTX-132 and P–T1000–exatecan inhibited tumor growth in growing tumors treated with Trodelvy (Fig. 3E; Supplementary Fig. S15B) and Dato–DXd (Supplementary Fig. S15C) or P–GGFG–DXd, respectively (Fig. 3F). In HCT-15, MTX-132 overcame the treatment ineffectiveness of Dato–DXd (Supplementary Fig. S15D). Together, T moiety–exatecan ADCs were effective in DXd/SN-38 ADC treatment–refractory tumors.

Following intravenous delivery at the same doses as the efficacy study (Supplementary Fig. S15E), pharmacodynamic response imaging measured by the DNA damaging marker γH2A.X was used to probe the PK profile of MTX-1000 in vivo. Although tumor size difference was insignificant in the first 3 days after ADC administration, the pharmacodynamic marker γH2A.X signal of MTX-1000 was significantly higher than DS-8201a on both days 1 and 3 (Supplementary Fig. S15F and S15G; Fig. 3G).

The improved intratumoral pharmacodynamic response exemplified by MTX-1000 translates into the antitumor effectiveness of T moiety–exatecan ADCs against large-size tumors. In xenograft models of starting tumor size of 500 to 1,000 mm³, MTX-1000 and MTX-132 induced tumor regression and were significantly more effective than DS-8201a and Dato-DXd/Trodelvy, respectively (Fig. 3H and I). Furthermore, MTX-1000 induced tumor regression in the DS-8201a–treated group when the tumor reached 1,300 mm³, whereas another dose of DS-8201a failed to inhibit tumor growth (Fig. 3H). The HER3-targeting ADC P–T1000–exatecan also inhibited tumor growth in tumors 800 mm³ and larger in ASPC-1, demonstrating significantly higher efficacy than P–GGFG–DXd (Supplementary Fig. S15H). The enhanced therapeutic index and improved intratumor pharmacodynamic response of T moiety–exatecan ADCs translate to improved antitumor efficacy in tumors with low target expression, tumors with DXd/SN-38 ADC treatment resistance, or large tumors, as summarized in the efficacy comparison of MTX-1000 versus DS-8201a as an example (Fig. 3J).

MDR transporters such as ABCG2 and P-gp confer chemotherapy resistance, a frequent cause of treatment failure, through active drug efflux. Previously, IMMU-132/Trodelvy was shown to overcome resistance to SN-38 in breast and gastric tumor models due to the combination of the ABCG2 inhibitor YHO-13351 with Trodelvy (17). The effect of MDR dependence of exatecan versus DXd/SN-38 ADCs targeting HER2, HER3, and TROP2 was investigated in representative endogenous ABCG2- and P-gp-high-expression cells (Supplementary Fig. S16A). ABCG2 inhibitor (YHO-13351) and P-gp inhibitor (tariquidar) improved the in vitro cytotoxicity potency of DXd ADCs or Trodelvy significantly but were less effective on IC₅₀ of T1000–exatecan ADCs (Supplementary Fig. S16B–S16D; Fig. 4A), which is largely consistent with payload data (Fig. 1D and G). In vivo, TROP2-targeting Trodelvy or Dato–GGFG–DXd alone was ineffective in HCT-15. When combined with the P-gp inhibitor tariquidar, both showed significant tumor inhibition (Fig. 4B). In comparison, Dato–T1000–exatecan alone was effective. A combination of Dato–T1000–exatecan with tariquidar was slightly more effective (Fig. 4B and C). As control, tariquidar alone or PBS was not effective (Fig. 4C). In HCT-15 and ASPC-1 (high ABCG2), DS-8201a alone was ineffective but led to significant antitumor activity in combination with tariquidar in HCT-15 (Fig. 4D) or the ABCG2 inhibitor YHO-13351 in ASPC-1 (Fig. 4E). MTX-1000 alone or in combination with respective inhibitor was equally effective in both models (Fig. 4D–F). Similar results were observed for the HER3-targeting ADC P–GGFG–DXd and counterpart P–T800–exatecan in HCT-15 and ASPC-1 (­Supplementary Fig. S16E; Fig. 4G). The efficacy improvement by YHO-13351 for P–GGFG–DXd in ASPC-1 was short-lived since the tumors started to grow again—even tumor growth in the P–T800–exatecan groups was kept inhibited (Fig. 4G). Finally, in a colon cancer PDX model with low TROP2 and high P-gp expression, tariquidar was able to improve Dato–GGFG–DXd efficacy significantly in large-size (600 mm³) tumor groups (Fig. 4H; Supplementary Fig. S16F). In this model, MTX-132 alone was effective for tumor inhibition (Supplementary Fig. S16F). These data suggest that a combination treatment of DXd/SN-38 ADC with a specific MDR inhibitor or T moiety–exatecan ADC alone was effective for MDR⁺ tumor resistance.

So far, ADCs have failed to demonstrate meaningful clinical efficacy against colon cancer. T moiety–exatecan ADCs targeting HER2, HER3, and TROP2 were evaluated in colon cancer PDX models representing unmet clinical needs (Fig. 5A). In a TP53–BRAF double mutation PDX model with high HER3 expression (IHC 3+), P–T1000–exatecan showed antitumor activity, but the tumors were resistant to P–GGFG–DXd (Fig. 5B). In a KRAS–TP53 double-mutant PDX model with HER2 (low expression 1+) and TROP2 (high expression 3+) positivity, T moiety–exatecan ADCs showed potent antitumor activity (Fig. 5C and D). For HER2, both MTX-1000 and MTX-800 showed significant tumor inhibition with one out of five achieving complete response (CR) in the MTX-1000 group (Fig. 5C). However, tumors were resistant to Tras–GGFG–DXd (Fig. 5D). TROP2-targeting Dato–GGFG–DXd showed comparable tumor inhibition to hRS7–T1000–exatecan (MTX-132) for 40 days before tumors in the Dato–GGFG–DXd treatment group grew again, leading to a significant efficacy gap between these two groups (Fig. 5D). In a TROP2-high-expression tumor with triple mutations of TP53, KRAS, and BRAF and microsatellite stability (MSS), MTX-132 showed more durable response than Dato–DXd (Fig. 5E). Together, T moiety ADCs were found to produce deep and durable antitumor response in colon cancer models resistant to current ADCs.

Antitumor agents targeting DDR pathway mediators, such as PARP and ATM/ATR/WEE1, have shown synergistic effects with ADCs (36, 37). A synergistic mechanism of combined TOP1 and CHEK1 inhibition was demonstrated in colon cancer (38). To explore the synthetic lethality of DXd or exatecan-based ADCs with DDR inhibitors, we first tested in vitro cytotoxicity of exatecan and DXd in combination with the PARP inhibitor talazoparib (Tala; Supplementary Fig. S17A) and a novel ATR inhibitor, M4344 (Supplementary Fig. S17B), at respective nontoxic doses (IC₂₀) for each inhibitor in COLO205 (39). Both DXd and exatecan show synergy with Tala and M4344, with an improvement of IC₅₀ 30- to 200-fold for Tala and 7- to 20-fold for M4344 (tabulated in Supplementary Fig. S17C and S17D). Tala improved the IC₅₀ of exatecan and DXd by roughly 200-fold, whereas M4344 did by only 10-fold (Supplementary Fig. S17E and S17F). Combining exatecan with Tala or M4344 was much more potent (10–60 times) than DXd plus Tala or M4344 (Supplementary Fig. S17G). Combination index (CI) score calculation suggested that exatecan synergized with both Tala and M4344 at a lower concentration than DXd and Tala synergized with both exatecan and DXd better than M4344 (Supplementary Fig. S17H and S17I). Cytotoxicity of MTX-1000 or Tras–GGFG–DXd was then tested with Tala or M4344. In COLO205, all molecules showed some activity in vitro (Supplementary Fig. S17J), and CI calculation showed that MTX-1000 synergized better with both Tala and M4344 compared with Tras–GGFG–DXd (Fig. 5F and G). In vivo, a combined use of MTX-1000 (3 mg/kg) with Tala produced significantly better tumor inhibition than MTX-1000 (effective), Tala (ineffective), or 10 mg/kg of Tras–GGFG–DXd with Tala (Fig. 5H). Even at 10 mg/kg, Tras–GGFG–DXd did not show synergy with Tala. Tras–GGFG–DXd combined with Tala inhibited tumor growth in a similar way to just Tras–GGFG–DXd. Similarly, MTX-1000 at 3 mg/kg was synergistic with M4344, and Tras–GGFG–DXd at 10 mg/kg was not (Fig. 5I). These data demonstrated that exatecan/MTX-1000 can synergize with DDR pathway mediators to achieve improved antitumor efficacy, outperforming DXd/Tras–GGFG–DXd.

We evaluated T moiety–exatecan ADCs targeting TROP2, HER2, and HER3 in lung cancer PDX models representative of unmet medical needs (Fig. 6A). Lung squamous cell cancer is a more challenging type of lung cancer to treat due to a lack of actionable mutations. MTX-132 caused significant tumor regression (CR in one model) in two models, whereas Dato–DXd was ineffective in one model and less effective in another (Fig. 6B and C). In a PDX model derived from an osimertinib (AZD9291; third-generation tyrosine kinase inhibitor)-resistant patient with EGFR del19/T790M/C797S (triple mutation) that lacked approved therapeutic options, combination therapy of the ALK inhibitor brigatinib with the anti-EGFR antibody cetuximab was able to induce tumor inhibition, which is consistent with a previous study (ref. 40; Supplementary Fig. S18A–S18D). Despite very low HER2 and HER3 expression in this model, MTX-1000 and P–T1000–exatecan produced significant tumor inhibition, with 3 of 5 and 1 of 5 mice, respectively, achieving CR (Fig. 6D and E). P–GGFG–DXd was ineffective in tumor inhibition, whereas Tras–GGFG–DXd initially produced three of five CR, but two tumors in this group started to regrow rapidly after 50 days compared with slight regrowth of two tumors in the MTX-1000 treatment group, leading to a significant difference between MTX-1000 and Tras–GGFG–DXd (Fig. 6D). In comparison, both MTX-1000 and P–T1000–exatecan significantly outperformed brigatinib combined with cetuximab to inhibit tumor growth in this model (Fig. 6F). Finally, in a PDX model with EGFR ex19del and MET amplification, P–T1000–exatecan produced CR in all 5 mice, whereas P–GGFG–DXd showed significant tumor inhibition in 3 mice, but no growth inhibition for the other two tumors, showing a significant difference between the efficacy of two ADCs (Fig. 6G and H; Supplementary Fig. S18E). This tumor was resistant to both erlotinib and osimertinib (Supplementary Fig. S18F).

Both microtubule-disrupting payloads and DXd-derived ADCs induce immunogenic cell death (ICD) and promote an antitumor response (41). We sought to determine whether MTX-1000 has an immunomodulatory effect. We first assayed exatecan's capability for inducing ICD through induction of endoplasmic reticulum stress and autophagy as well as involving calreticulin (CRT) cell-surface exposure. In the exatecan-sensitive cell line COLO205, treatment with exatecan and MTX-1000 led to a significant externalization and plasma membrane exposure of CRT (Supplementary Fig. S19A and S19B), suggesting that exatecan and released exatecan from MTX-1000 through target-dependent internalization of MTX-1000 induced strong ICD. The increases in relative mean fluorescence intensity (rMFI) and percentage of positive cells of surface CRT for exatecan/MTX-1000 were significantly higher than that of DXd/T–GGFG–DXd's (Supplementary Fig. S19A and S19B), suggesting that exatecan/MTX-1000 may have better immunogenic properties. The enhanced CRT was observed despite 30% to 70% decreases in cell number after 48 hours (Supplementary Fig. S19C), compared with controls.

To translate the in vitro exatecan/MTX-1000–induced ICD phenotype into enhanced antitumor activity in vivo, we generated a syngeneic mouse model using CT-26 with transfected human HER2 (CT26-hHER2). CT26-hHER2 had medium (rMFI of 30–50) hHER2 expression determined by flow cytometry (Supplementary Fig. S19D). The combination of MTX-1000 and Tras–GGFG–DXd with an anti–PD-1 antibody was examined. At day 12, all experimental groups showed significant tumor inhibition compared with the vehicle control (Supplementary Fig. S19E). Similar to other models, monotherapy of MTX-1000 (10 mg/kg, once a week, twice) showed better tumor inhibition than Tras–GGFG–DXd (**, P = 0.09), with a TGI of 61% compared with the latter's 38% (Supplementary Fig. S19F). Interestingly, monotherapy of MTX-1000 was similarly effective as a combination therapy of Tras–GGFG–DXd with PD-1 (not significant), with a TGI of 61% and 62%. Combination efficacy of MTX-1000 with PD-1 (TGI = 78%) was significantly higher than PD-1 alone (**, P = 0.002; TGI = 45%) than to MTX-1000 alone (*, P = 0.0179; TGI = 61%), suggesting that MTX-1000 might play a more prominent role in the improved efficacy of combination therapy (P = 0.002). The combined use of MTX-1000 and anti–PD-1 antibody produced an antitumor effect that was superior to both monotherapies (Supplementary Fig. S19F, right). No body weight abnormalities were observed in mice (Supplementary Fig. S19G). The survival rates for vehicle, Tras–GGFG–DXd, anti–PD-1 antibody, MTX-1000, Tras–GGFG–DXd + PD-1, and MTX-1000 + PD-1 at day 21 were 0/8, 1/8, 2/8, 3/8, 4/8, and 7/8, respectively. TGI of MTX-1000 combined with PD-1 was significantly better (*, P = 0.0195) than that of Tras–GGFG–DXd combined with PD-1 (Supplementary Fig. S19F tabulation).

Camptothecin class TOP1 inhibitors have become more prominent as ADC payloads thanks to the clinical success of DS-8201a and Trodelvy (30, 42–44). The characterization of novel camptothecins largely focuses on the structure–activity relationship, and the scope of preclinical ADC studies is limited to routine efficacy and toxicity examination (23, 30, 42, 43). However, the development of an optimal ADC necessitates a more holistic approach in selecting linker chemistry and the incorporation position of a hydrophilic polymer moiety (often required) within the ADC architecture (30, 45–47). In order to improve DXd/SN-38 ADC performance, an ADC with high clinical translatability will need to be developed, which will warrant a thorough mechanistic investigation of the new TOP1 inhibitor–based ADC design.

Here we present our effort to develop a class of exatecan ADC with such clinical potential. Exatecan was selected as the payload due to its higher potency, known clinical and toxicity profile in humans, and lower MDR-mediated efflux and higher cell permeability compared with DXd/SN-38 (48). Mechanistic differences in the biochemical, structural, and pharmacologic profiles of exatecan and DXd/SN-38 were investigated. Exatecan was shown to have a 10-fold higher potency and 1/10th efflux ratio in MDR⁺ cells, two traits that correspond with higher efficacy and the ability to overcome resistance toward low-target-expression and MDR⁺ tumors. Higher cell permeability and stronger bystander penetration of exatecan may further enhance its ADC function (21). On the other hand, a higher potency could imply more severe side effects. Therefore, exatecan's linker chemistry and the conjugation strategy for exatecan must incorporate its superior pharmacologic feature while maintaining or improving upon safety in the final ADC. To achieve these objectives, we focused on the physicochemical evaluation of distinctive ADC designs to compare and select a novel chemistry architecture, T moiety. The modification of pAB adjacent to exatecan not only led to a highly homogeneous and hydrophilic ADC through efficient masking of payload hydrophobicity, but also created a simple, ready process to test established linker structure without additional chemistry. The selection of a VA linker in combination with pAB modifications by pSAR (T1000; or a minimal m-methylaminomethyl group, T800) resulted in more hydrophilic, less aggregated, and more stable antibody–exatecan conjugates than alternative linkers (GGFG or VC), modification site (linker/MC), or modification chemistry (PEG), which was consistent with previous observations and principles (30, 45–47). T moiety chemistry was validated by the successful translation of higher potency and less MDR sensitivity of exatecan into corresponding ADCs. Performance improvement of T moiety–exatecan ADC was mediated by exatecan through stronger TOP1 inhibition, lower MDR-mediated efflux of released payload, and a deeper and broader intratumoral pharmacodynamic response. The mechanistic understanding of T moiety–exatecan ADC may provide a broader implication for designing new TOP1 ADCs. The consistent high-quality antitumor response of T moiety–exatecan ADCs in relevant PDXs and organoids with a high patient response predictability bodes well for clinical translation (34, 49).

Low-target-expression and MDR⁺ tumors represent important resistance mechanisms for ADC (8, 9). We demonstrated here that DXd ADC was susceptible to similar resistance. However, T moiety–exatecan ADCs showed less resistance and often displayed potent and durable antitumor activity in low-target-expression and MDR⁺ tumors, compared with a combination treatment of ABCG2/p-pg inhibitor and DXd/SN-38 ADCs that was shown to be effective but less persistent. Although MDR inhibitors, including ABCG2 inhibitor (YHO-13351) and P-gp inhibitor (tariquidar), are commonly used in ADC studies (17), the mechanism of the observed sensitivity difference among various TOP1 inhibitors remains to be clarified in the future due to the complexity of MDR biology (50). Due to potential selectivity issues of MDR inhibitors, there remains the possibility of other substrates of the inhibitors contributing to the overall cytotoxicity differences. Nevertheless, enhanced therapeutic indices of T moiety–exatecan ADCs targeting classic tumor antigens such as HER2/TROP2/HER3 may provide a new opportunity to treat target-positive patient populations beyond the reach of current ADCs. For example, T moiety–exatecan ADCs generated durable and deep responses in resistant or treatment-refractory colon cancer models. In colon cancer cells, MTX-1000 showed highly synergistic antitumor efficacy with PARP/ATR inhibitors, suggesting that a combination therapy strategy of exatecan-based ADCs with DDR pathway inhibitors could augment antitumor potency (30, 38). Similarly, T moiety–exatecan ADCs showed improved efficacy against lung cancer intrinsic resistance mechanisms. Although the degree of improvements varied among tumor models, likely due to a combination of factors such as target antigen expression level, tumor cell sensitivity toward payload, degree of bystander effect, and tumor microenvironment, T moiety ADCs showed greater efficacy gain in more challenging models. One possible explanation was that the combined mechanistic improvements of T moiety–exatecan ADCs resulted in an elevated cumulated payload inside the tumor cells beyond the functional inhibition threshold, thus yielding more qualitative differences in these models. More preclinical data and eventually clinical data will help establish the most responsive tumor types for each class of ADC.

T moiety–exatecan ADCs maintained a higher potency without increasing toxicity with HNSTDs in nonhuman primate studies at a level that was similar to or higher than that of other TOP1 inhibitor ADCs. Notably, side effects from the administration of T moiety–exatecan ADC were often more prominently observed in the GI tract (diarrhea) and the hematologic system (reticulocyte reduction), though symptoms in both were fully reversible. The hematologic and digestive organ toxicities for T moiety–exatecan ADCs mirrored those of exatecan as a single agent in human trials (48). However, T moiety–exatecan ADC achieved more favorable safety signals in the GI tract and minimal myelotoxicity than exatecan, possibly reflecting the ADC design's superior physicochemical features. Improved stability, longer ADC half-life, lower free payload release, and the reduced myelotoxicity potential of ADC in comparison with free payload may all contribute to the favorable toxicity profile of T moiety–exatecan ADC. Interstitial lung disease occurred in just over 10% of DXd-based ADC–treated patients during clinical trials. Although we did not observe any structural damage or signs of inflammation in the lungs of animals dosed with T moiety–exatecan ADCs, it remains uncertain whether prolonged treatment will yield additional pulmonary toxicity. Nevertheless, it is conceivable that T ­moiety–exatecan ADCs could have different target-independent tissue distributions than DXd-based ADCs, resulting in the manifestation of diverse symptoms.

For tumors less responsive or resistant to TOP1 inhibitors, payload classes with different mechanisms may be necessary. T moiety is a modular structure compatible with diverse linker chemistry and is amenable to building ADCs with dual payloads. Taken together, T moiety–exatecan ADCs have the potential to address patient needs unfulfilled by current ADCs, whereas the versatility and scalability of T moiety can facilitate the introduction of payloads of completely different MOAs to meet the continuous challenges of drug resistance.

Synthesis of T moiety and all other chemical compounds is described in the Supplementary Note.

The anti-HER2 antibody (Tras) was purchased from Geneway Biotechnology Co., Ltd. The anti-CDH6 antibody (hG019), anti-TROP2 antibodies (Dato and sacituzumab), and anti-HER3 antibody (patritumab) were all generated in the CHO or 293F expression system and purified by Mabselect Sure Resin (GE Healthcare) in gravity column. The sequences of these antibodies were obtained from the corresponding patents and literature. T-DM1 was purchased from F. Hoffmann-La Roche (Basel, Switzerland). Control IgG1 was purchased from Beijing Solarbio Science and Technology Co., Ltd. Anti-mouse PD-1 antibody is produced by ChemPartner (batch#: 201630002). ADCs were synthesized as described above, and their DAR values were determined by reverse-phase chromatography. The drug distribution was analyzed by HIC and further characterized by reduced reverse-phase LC/MS (Supplementary Note). T-DXd/DS-8201a was made in-house with Tras (Batch#: FX03DS1901, Geneway Biotech) using linker–payload directly purchased from MedChemExpress: MC-GGFG–DXd (HY-114233, MedChemExpress). Talazoparib (HY-108413), exatecan (HY-13631), DXd (HY-13631E), and M4344 (HY-136270) were all purchased from MedChemExpress.

A solution of naked mAb (10 mg/mL in PBS (7.0) + 2 mmol/L EDTA) was treated with 7 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) for 2 hours at 37°C. Then 14 molar equivalents of the linker drug (from a 10 mmol/L DMA stock solution) were added to the fully reduced antibody while keeping residual DMA concentration below 10% (v/v). The mixture was incubated at room temperature for an hour followed by purification over a Zeba spin desalting column (Thermo Fisher Scientific) to remove excess reagents. In this step, the resulting ADC was also buffer exchanged into formulation buffer (20 mmol/L histidine, 150 mmol/L NaCl, pH 5.5). Moreover, the excess linker drug could be removed thoroughly by treating the product with activated charcoal (30 mg of charcoal to 1 mL ADC solution) followed by vortexing for 2 hours at room temperature. The charcoal was then removed via sterile filtration (0.2 μm PES filters), and the final ADC was stored at −80°C.

The concentration of mAb and the conjugated drug in the ADC was calculated by measuring UV absorbance of an aqueous solution of the ADC at two wavelengths of 280 nm and 370 nm, as the total absorbance at any given wavelength is equal to the sum of the absorbance of all light-absorbing chemical species that are present in a system (additivity of absorbance). The equations were as follows:

The values of ε_A,280, ε_A,370, ε_D,280, and ε_D,370 were estimated based on calculation of the amino acid sequence of the antibody or obtained by UV measurement of the compound according to the Lambert–Beer law. Simultaneous equations (1) and (2) can be solved by substitution of the above values, and then C_A and C_D were determined. Dividing C_D by C_A, the average number of conjugated drug molecules per antibody was determined.

Aggregation was assessed by size exclusion chromatography, which was performed on an Agilent 1260 Infinity II HPLC system with UV detection at 280 nm. The column was a TSKgel G3000SW_XL column (Tosoh Bioscience). Samples were injected at a 50 μg load. The mobile phase was 200 mmol/L sodium phosphate and 150 mmol/L sodium chloride (pH 7.0). Also, 15% (v/v) isopropanol was added to the mobile phase to minimize secondary hydrophobic interactions with the stationary phase and prevent bacterial growth. The flow rate was 0.75 mL/minute, and the column temperature was set at room temperature.

ADCs with different numbers of drugs per antibody were separated using a butyl HIC column (TSK-gel Butyl-NPR 4.6 × 35 mm 2.5 μm, Tosoh Bioscience) at room temperature. HIC was also performed on an Agilent 1260 Infinity II HPLC system with UV detection at 280 nm. Mobile phase A was 1.5 mol/L (NH₄)₂SO₄, 50 mmol/L K₂HPO₄, pH 7.0, and mobile phase B was 21.3 mmol/L KH₂PO₄, 28.6 mmol/L K₂HPO₄, 25% (v/v) isopropanol, pH 7.0. The gradient program was as follows: B %: 0% to 25% (0–1 minutes, 0.8 mL/minute), 25% (1–3 minutes, 0.6 mL/minute), 25% to 80% (3–13 minutes 0.6 mL/minute), 80% (13–17 minutes, 0.6 mL/minute), 80% to 0% (17–17.10 minutes, 0.5 mL/minute), and 0% (17.10–25 minutes, 0.7 mL/minute).

For cell-surface proteins, IF and FACS assays were performed under nonpermeable conditions without detergent in the buffers. Antibody binding signal was detected using Alexa Fluor 488 and 594 goat anti-mouse IgG secondary antibodies (Jackson ImmunoResearch, no. 115-545-003 and no. 115-585-003). Briefly, cells attached on coverslips (IF assays) or suspended in 1× PBS (FACS assays) were first fixed in 4% paraformaldehyde (PFA) for 10 minutes. PFA was then removed, and cells were rinsed 3 times with 1× PBS. Cells were blocked overnight at 4°C in blocking buffer (1× PBS containing 10% normal goat serum; 0.1% Triton was added for intracellular proteins). After removing the blocking buffer, cells were incubated with primary antibody (dilution in the blocking buffer at 1:100 to 1,000) for 3 hours at room temperature. Cells were rinsed 6 times in 1× PBS before being incubated with fluorescence-labeled secondary antibody (diluted in blocking buffer at 1:500 dilution ratio with 1:10,000 Hoechst 33258; Sigma, no. 94403) for 1 hour. Last, cells were rinsed 3 times with 1× PBS. IF images were recorded with a Nikon confocal system A1Si. The 3D reconstruction of the IF results was performed in ImageJ (NCBI, NIH free software). The FACS data were collected using a BD Accuri C6 Plus system. A control sample without a primary antibody and another sample with an isotype control antibody were used. The LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) was used to exclude dead cells.

COLO205 (RRID:CVCL_0218), HCT-15 (RRID:CVCL_0292), and DLD-1 (RRID: CVCL_0248) cell lines were purchased from the ATCC in 2020. HT-29 (RRID:CVCL_0320), SK-MES-1 (RRID:CVCL_0630), NCI-H292 (RRID:CVCL_0455), MDA-MB-468 (RRID:CVCL_0419), SK-OV-3 (RRID:CVCL_0532), LoVo (RRID:CVCL_0399), NCI-H460 (RRID:CVCL_0459), BxPC-3 (RRID:CVCL_0186), and PC9 (RRID:CVCL_B260) cell lines were purchased from a stem cell bank, Chinese Academy of Sciences (Shanghai, China), in 2018. Cells were maintained in RPMI 1640 medium (COLO205, NCI-H292, HCT-15, DLD-1, NCI-H460, BxPC-3, and PC9) or DMEM (SK-MES-1 and MDA-MB-468) or F-12K medium (LoVo) or McCoy's 5A medium (HT-29 and SK-OV-3) supplemented with 10% heat-inactivated FBS and cultured at 37°C in a 5% CO₂ atmosphere. When cells were grown to ∼80% confluence, they were dissociated from culture plates by treatment with 1× PBS and 1 mmol/L EDTA for 10 to 20 minutes. Trypsin was not used to avoid damage on cell-surface proteins. rMFI was calculated by the ratio of MFI of antitarget Ab-FITC/MFI of isotype control-FITC.

The mouse colon cancer cell line CT26 (RRID:CVCL_7254) was purchased from the ATCC in 2020. An empty vector (pQCXIN; Clontech) or human HER2 gene (sequence based on NM_004448.3) was retrovirally introduced into CT26 cells (CT26 and CT26-hHER2, respectively). The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 250 μg/mL geneticin (Thermo Fisher Scientific) at 37°C in a 5% CO₂ atmosphere. Human HER2 expression was confirmed by flow cytometry.

All PDX models (362014, 361795, 360837, 200717, 200662, 200615, and 360961) were obtained from Lidebiotech Co., Ltd. in 2021.

All cell lines were authenticated by short tandem repeat analysis according to suppliers’ information. All cells were tested negative for Mycoplasma contamination using the Mycoplasma PCR Detection Kit (MP004, GeneCopoeia) prior to conducting cell-based assays. All cells and PDX models were used for the experiments within 5 to 15 passages and 2 to 5 passages, respectively, after collection.

Cells were seeded to a 96-well plate at an appropriate density depending on the cell line (typically 1,000–3,000 cells per well in 100 μL of appropriate culture media). After overnight incubation, a concentration series of small-molecule or ADC drugs and controls was added. Cell viability was evaluated after 5 or 6 days. MTT (5 mg/mL, 20 μL, Sigma-Aldrich) was added into the wells, and incubation was continued for 2 to 4 hours at 37°C. Culture media were then removed, and well content was homogeneously dissolved with 0.1 N HCl/isopropanol. Absorbance values were measured on a Thermo Scientific Multiskan EX microplate reader using a wavelength of 570 nm (with a reference wavelength of 690 nm). The IC₅₀ values compared with untreated cells or treated with other controls were determined using inhibition dose–response curve fitting (GraphPad Prism 9). Target expression on each cell line was measured by flow cytometry as described above.

All tumor-bearing models were established in female BALB/c nude mice that were purchased from the Shanghai Family Planning Research Institute. All in vivo studies were performed in accordance with the local guidelines of the Institutional Animal Care and Use Committee of Multitude Therapeutics and with the approval of the committee. Briefly, 4- to 6-week-old mice were housed together in sterilized cages and maintained under required pathogen-free conditions. Each cell suspension or tumor fragment was inoculated subcutaneously into female nude mice. When the tumor had grown to an appropriate volume, the tumor-bearing mice were randomized into treatment and control groups based on the tumor volumes, and dosing was started. Antibodies and ADCs were administered intravenously to the mice. Small-molecule drugs were administered orally. Tumor volume defined as 1/2 × length × width² was measured twice (3–4 days) a week. TGI (%) was calculated as follows: TGI (%) = [1 – (mean of treatment group tumor volume on evaluation day)/(mean of control group tumor volume on evaluation day)] × 100. The mice were euthanized with CO₂ gas when they reached endpoints (tumor volume exceeding 3,000 mm³, >10% reduction of body weight, or clinical signs indicating that mice should be euthanized for ethical reasons).

Female BALB/c mice, weighing approximately 18 to 20 g, were purchased from Shanghai Lingchang Biotech Co., Ltd. and were maintained with 5 mice per cage in an individually ventilated cage in a specific pathogen–free animal facility at ChemPartner with autoclaved cages. After 9 days of acclimation, animals were subcutaneously inoculated with CT26-hHER2 cells (3 × 10⁵ cells/0.1 mL/mouse) on the right flank for tumor development. Ten days after inoculation, 36 mice with tumor sizes ranging from 100 to 200 mm³ (average tumor size 178 mm³) were enrolled into the efficacy study and randomized into six therapy groups, and the treatments were initiated on the randomization day (defined as D0). MTX-1000, DS-8201a (10 mg/kg), and anti–PD-1 antibody (5 mg/kg) were administered intravenously at a volume of 10 mL/kg to mice. As a control, ABS buffer (10 mmol/L acetate buffer, 5% sorbitol, pH 5.5) was administered at the same volume as the drugs. MTX-1000 and DS-8201a were administered on days 0 and 7. Anti–PD-1 antibody was administered on days 0, 3, 7, and 10.

For flow-cytometric analysis of T cells, dendritic cells, and tumor cells, when the average volume of tumors reached approximately 250 to 400 mm³ (8 days after tumor inoculation), the mice were treated with vehicle or DS-8201a (10 mg/kg, once, intravenously; day 0). The mice were euthanized with CO₂ asphyxiation on day 8, and tumors were cut into small pieces and dissociated with the Tumor Dissociation Kit (Miltenyi Biotec) by the gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec). The resultant single cells were blocked with Mouse BD Fc Block reagent (Becton Dickinson) and stained with antibodies against mouse CD3, CD4, CD8, CD11c, CD45, CD86, granzyme B, MHC class I, MHC class II, and PD-L1 and human HER2.

Sections from formalin-fixed, paraffin-embedded tumor tissue blocks were stained using Opal multiplex according to the manufacturer's protocol (PerkinElmer) for DAPI, CD45, CD4, and CD8. Slide scanning was performed on the Vectra 3.0 instrument. All the images were analyzed with the HALOTM Image Analysis platform. The whole section was analyzed, and the big necrosis area and stroma area were excluded. Single-positive cells were counted separately. Images were spectrally unmixed, evaluated for staining intensities and morphology, tissue segmented based on tissue markers, cell segmented based on nuclear and membrane markers, and phenotypically scored.

Concentrations of ADC and the total antibody in plasma were determined with a validated ligand-binding assay; the lower limit of quantitation was 0.02 μg/mL. Briefly, immunoplates were coated with 1 μg/mL Human HER2 Protein, His Tag (Acrobiosystems Inc., Beijing) in coating buffer and kept overnight at 4°C. After washing, the plates were blocked, and each serially diluted sample was added to the wells. After incubation for 1 to 2 hours at 37°C, the plates were washed and incubated with HRP-conjugated anti-human IgG Fc secondary antibody (Abcam Inc.) for total antibody measurement or a biotin-labeled anti-exatecan/DXd antibody (Abmart) for ADC measurement. After reaction at 37°C for 1 to 2 hours, TMB solution (KPL Inc.) was added directly or after incubation with Streptavidin Protein, HRP (Thermo Scientific) for 40 to 60 minutes at 37°C. A₄₅₀ in each well was measured with a microplate reader. Concentrations of payload exatecan/DXd in plasma were determined with a validated LC/MS-MS method; the lower limit of quantitation was 0.05 ng/mL. MTX-1000/DS-8201a was intravenously administered at 4.0 mg/kg to rats. Plasma concentrations of ADC, total antibody, and DXd were measured up to 21 days after dose.

The release rate of exatecan/DXd from MTX-1000/DS-8201a at the concentration of 10 mg/mL at 37°C up to 21 days was evaluated in mouse, rat, monkey, and human plasma.

For a binding assay, immune plates were coated with 2.5 mg/mL His-tagged HER2-ECD protein (Sino Biological) in coating buffer and kept overnight at 4°C. After washing, the plates were blocked, and each serially diluted substance was added to the wells. After incubation for 1.5 hours at 37°C, the plates were washed and incubated with HRP-conjugated anti-human IgG secondary antibody for 1 hour at 37°C. After washing, TMB solution was added and A450 in each well was measured with a microplate reader. For the detection of phosphorylated Akt (pAkt), SK-BR-3 cells were preincubated in a 96-well plate for 4 days and then incubated with each substance for 24 hours. After incubation, the cells were lysed and intercellular pAkt and total Akt were detected using a PathScan Phospho-Akt1 (Ser473) Sandwich ELISA Kit and PathScan Total-Akt1 Sandwich ELISA Kit (Cell Signaling Technology) according to the manufacturer's instructions. The relative pAkt of each sample well was calculated by dividing treated normalized pAkt values by untreated normalized pAkt values.

KPL-4 cells were treated with each substance. After 24, 48, or 72 hours, the cells were harvested and lysed with M-PER lysis buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The samples were loaded and separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The membranes were blocked and probed overnight with anti–phospho-Chk1 (Ser345; 133D3) rabbit mAb, anti-Chk1 (2G1D5) mouse mAb, anti–cleaved PARP (Asp214) antibody, anti–β-actin (8H10D10) mouse mAb (Cell Signaling Technology), anti–phospho-Histone H2A.X (Ser139) antibody (Millipore), and anti–Histone H2A.X antibody (Abcam) at 4°C. Then, the membranes were washed and incubated with fluorescence-labeled secondary antibodies for 10 minutes using SNAP intradermally (Millipore). The fluorescence signal was detected using an Odyssey imaging system (LI-COR, Inc.).

DNA-trapped TOP1 was determined by a modified RADAR assay following a previous study (19). After treatment with TOP1 inhibitors, COLO205 cells (1–3 × 10⁶ cells/sample) were washed with 1× PBS and lysed with 600 μL of DNAzol (Invitrogen) followed by precipitation with 300 μL of 200-proof ethanol by centrifugation at 14,000 rpm. The nucleic acids were collected, washed with 75% ethanol, resuspended in 200 μL of TE buffer, and then heated at 65°C for 15 minutes followed by shearing with sonication (40% output for 10-second pulse and 10-second rest repeated 4 times). The samples were centrifuged at 15,000 rpm for 5 minutes at 4°C, and the supernatant was collected. The sample (1 μL) was saved for the spectrophotometric measurement of absorbance at 260 nm to quantitate DNA content (NanoDrop). Two milligrams of each sample were subjected to slot blot for immunoblotting with anti-TOP1 antibody (no. 556597, BD Biosciences) or anti-dsDNA antibody (no. 3519, Abcam) as a loading control. The intensity of TOP1 and DNA was quantified by densitometric analysis using ImageJ.

MTX-1000 was intravenously administered intermittently at 3-week intervals over a 6-week period to cynomolgus monkeys (Table 1). Clinical signs, body weight, food consumption, and clinical pathology were monitored throughout the study. A necropsy was conducted on the day after the last administration. The reversibility of the toxic changes was assessed in a subsequent 6-week recovery period in cynomolgus monkeys.

Human colon cancer organoids were prepared as previously described (34). Organoids were harvested and seeded in a 96-well cell culture plate (Corning, 3799) when organoids grew to 50 μm in diameter. The sandwich method was used for drug tests. Before seeding, 50 μL 50% Matrigel (Corning, 356231, Matrigel mixed with PBS 1:1) was dropped on the bottom of the culture plate as the bottom layer. Then, 10 μL 10% Matrigel (Matrigel mixed with PBS 1:9) containing 50 ± 20 organoids was dropped on the bottom layer as the middle layer. Organoid culture medium (200 μL) was added in each well as the upper layer. Drug tests would start after 1 day of culture.

For drug tests, calcein AM was used for living cell staining at day 0. RCO culture medium was removed and replaced by a 200 μL drug-containing culture medium. The drug-containing culture medium was refreshed again after 3 days. Then, calcein AM/PI was used for living/dead cell staining at day 6. Z-stack imaging technique was used for the photographing of organoids at days 0 and 6 (Nikon, Ti2-E). The size of alive organoids was measured using NIS-Elements AR software (Nikon).

All statistical analyses except for toxicity studies were performed using SAS System Release 9.1.3 and 9.2 (SAS Institute Inc.). Statistical analysis in toxicity studies was performed using MUSCOT (YukmsCo., Ltd.). All IC₅₀ and ED₅₀ values were determined by a Sigmoid Emax model, and dose dependency was evaluated by a Spearman rank correlation coefficient hypothesis test.

Tests to determine significance are detailed in the relevant figure legends. Briefly, t tests or one-way ANOVAs were used to determine the statistical significance of treatment conditions and two-way ANOVA to discriminate between the contributions of both dose and transformation.

Correspondence and requests for data underlying the findings or materials should be addressed to Xun Meng ([email protected]).

S.-H. Liu reports other support from Multitude Therapeutics outside the submitted work. No disclosures were reported by the other authors.

W. Weng: Resources, investigation, visualization, methodology. T. Meng: Conceptualization, resources, formal analysis, investigation, writing–review and editing. Q. Zhao: Resources, investigation, visualization, methodology. Y. Shen: Investigation, visualization. G. Fu: Investigation, visualization. J. Shi: Investigation, visualization. Y. Zhang: Validation, investigation, visualization. Z. Wang: Investigation, visualization. M. Wang: Resources, validation, investigation, visualization. R. Pan: Investigation, visualization. L. Ma: Investigation, visualization. C. Chen: Investigation, visualization. L. Wang: Investigation, visualization. B. Zhou: Investigation, visualization. H. Zhang: Resources, investigation, visualization. J. Pu: Investigation, visualization. J. Zhang: Investigation, visualization. Y.P. Hu: Investigation, visualization. G. Hua: Conceptualization, resources, project administration, writing–review and editing. Y. Qian: Investigation, visualization. S.-H. Liu: Validation, investigation, visualization, writing–original draft, writing–review and editing. W. Hu: Validation, investigation, visualization, writing–original draft, writing–review and editing. X. Meng: Conceptualization, resources, formal analysis, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing.

This study was funded by Multitude Therapeutics, Abmart Inc., HySlink, Mabcare, D1 Medical Technology Company, Guangdong Provincial Key Laboratory of Chiral Molecule and Drug Discovery (2019B030301005), and Fudan University Shanghai Cancer Center. We thank Y. Tang of Multitude Therapeutics for initiating this project and for providing encouragement and support throughout the project. We thank X.X. Meng for reading and editing the manuscript. Finally, we thank Professor Hongbin Ji at the State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China, for encouragement and helpful suggestions.

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