Supplementary Materials and Methods

Characterization of MW282 mAb, parental mAb of 9MW2821.

Mouse body weight from MDA-MB-468, NCI-H322 and HT1376 cell-derived xenograft studies.

Binding affinity of MW282 mAb, 9MW2821, and enfortumab vedotin based on enzyme linked immunosorbent assay

Analytical results of MW282 mAb and 9MW2821

Cadherin 17 (CDH17), a member of the 7D-cadherin superfamily, is critically involved in intercellular adhesion, tissue integrity, and intestinal peptide transport. It is predominantly expressed on the basolateral membrane of intestinal and pancreatic ductal epithelial cells, with minimal expression in non-gastrointestinal (GI) tissues. Overexpression of CDH17 is strongly associated with tumor progression and metastasis in colorectal, gastric, and pancreatic cancers, making it an attractive target for therapeutic intervention. Here, we report the development of 7MW4911, a novel ADC designed to selectively target CDH17-positive tumors while sparing normal tissues. CDH17 expression as a therapeutic target was validated using comprehensive omics analyses. An anti-CDH17 specific mAb Mab0727 was generated using Mabwell’s integrated novel antibody discovery system. The ADC 7MW4911 was constructed by conjugating Mab0727 to the proprietary payload MF-6 via a cleavable linker, achieving a drug-to-antibody ratio (DAR) of 4 using site-specific inter-chain disulfide bond conjugation technology. Binding affinity, internalization, and cytotoxicity of 7MW4911 were evaluated in CDH17-positive GI cancer cell lines. Efficacy was further assessed in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models. Pharmacokinetics and safety were studied in cynomolgus monkeys. Immunohistochemistry (IHC) analysis confirmed CDH17 as a highly specific target, with expression observed in 100% (110/110) of colorectal, 77% (110/142) of gastric, and 55% (58/105) of pancreatic cancer samples. 7MW4911 demonstrated strong binding, efficient internalization, and potent cytotoxicity in CDH17-positive cell lines. In vivo, 7MW4911 showed substantial tumor growth inhibition (TGI), with TGI ranging from 29-97% in nine colorectal CDX models, 56-95% in three gastric models, and 81% in one pancreatic model. In PDX models, TGI ranged from 71-99% across nine colorectal and one gastric model. Importantly, 7MW4911 outperformed MMAE-based ADCs in multidrug-resistant CDX and PDX models. Pharmacokinetic studies in cynomolgus monkeys demonstrated a favorable safety profile, with a maximum tolerated dose exceeding 20 mg/kg and minimal off-target toxicity. 7MW4911 demonstrated exceptional preclinical efficacy, including robust activity against multidrug-resistant cancers, along with a strong safety profile. These findings highlight its potential as a transformative therapeutic candidate for CDH17-positive GI cancers, addressing significant unmet needs in oncology. Rui Wang, Peng Fang, Wei Zhou, Cuicui Guo, Xiaoding Tan, Hai Wu, Xun Gui. 7MW4911, a novel cadherin 17-targeting ADC, demonstrates potent efficacy in preclinical models of gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5466.

Summary of pharmacokinetics studies in 9MW2821 and enfortumab vedotin in tumor-bearing mice

B7-H3, an attractive target for antibody drug conjugate, is overexpressed in multiple tumors. Overexpression of B7-H3 in tumor cells frequently correlates with fewer tumor-infiltrating lymphocytes, faster cancer progression, and poor clinical outcome in several malignancies. Preliminary evidence revealed PARP inhibitors (PARPi) and B7-H3-directed antibody-drug conjugate (ADC) as two practical approaches to overcome chemoresistance in solid cancers. Thus, synthetic lethality exploitation by an anti-B7-H3 ADC plus PARPi might be a promising strategy in solid tumor models. Here, we investigated the activity of 7MW3711, a B7-H3-directed ADC bearing a novel isomerase I inhibitor (TOP1i) payload, alone and in combination with PARPi, in preclinical models to evaluate the potential for future clinical application. 7MW3711 was evaluated in vitro and vivo efficacy with or without PARPi (Olaparib or AZD5305). In vitro, human cancer cell lines were treated with 7MW3711 and two PARPi to determine the effect on anti-proliferative effects, double-stranded DNA breaks, and cell-cycle arrest. In vivo, mice bearing ovarian and SCLC tumor xenografts were treated with the combination of 7MW3711 and PARPi. Evaluation of the drug resistance ability in cell lines expressing P-gp revealed 7MW3711 exhibited > 3-fold more potent than DS7300 (B7-H3-directed DXd ADC), indicating that 7MW3711 has a lower efflux efficiency in drug-resistant strains. 7MW3711 combined use of PARPi synergistically potentiated the inhibitory effects of 7MW3711 on the growth of B7-H3-positive cancer cells, inducing DNA damage and apoptosis. The highest level of double-stranded DNA breaks was observed in cells treated with combination which can be determined in the upregulation of γ-H2AX protein. Combination increased both G2/M phase-arrested cells and S-phase cells, reduced G1-phase, indicating that most proliferating cells were arrested. Combining 7MW3711 with olaparib or AZD5305 produced significant antitumor effects and survival benefit above that achieved with monotherapy in various B7-H3-positive solid tumor xenografts. Collectively, our results demonstrate the synergistic antitumor activity of combining 7MW3711 with PARPi, suggesting that DNA damage-repair inhibitors in combination with B7-H3-targeted ADCs is a promising approach for treating B7-H3-positive malignancies. These data provide evidence for the potential utility of 7MW3711 combination with PARPi for treatment of B7-H3-expressing tumors and support the rationale for further clinical studies. Xiaowei Cen, Peng Fang, Yueyue Yang, Dongan Yu, Yueqin Yao, Fei Mei, Xueying Li, Meng You, Yiyan Zhang, Long Yin, Xiaowei Sun, Xiaoding Tan, Wei Zhou, Hai Wu. Combined a B7-H3-targeting antibody-drug conjugate, 7MW3711, and PARP inhibitors synergistically potentiates the antitumor activity in B7-H3-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 830.

Mouse body weight from BL3578, CV13641, LU3073, BR9457, BR9479, BR1282 and BR1458 patient-derived xenograft studies.

Cadherin 17 (CDH17) has emerged as a promising target for gastrointestinal (GI) cancers, which are often complicated by multidrug resistance (MDR) and recurrence. In this study, we developed 7MW4911, a CDH17-targeted antibody-drug conjugate (ADC) that incorporates a topoisomerase inhibitor MF-6 (Topi MF-6) payload linked via a cleavable linker, designed specifically to address MDR in GI cancers. 7MW4911 exhibited high specificity for CDH17-expressing cancer cells and potent cytotoxicity in vitro. In preclinical models, including patient-derived xenografts (PDXs) with distinct mutations, 7MW4911 achieved tumor growth inhibition ranging from 71% to 99%. Remarkably, 7MW4911 outperformed monomethyl auristatin E (MMAE)-based and Deruxtecan (DXd)-based ADCs in MDR models, highlighting its effectiveness against drug-resistant cancer phenotypes. Additionally, 7MW4911 showed favorable pharmacokinetics and a highest non-severely toxic dose (HNSTD) exceeding 20 mg/kg in cynomolgus monkeys, underscoring its promising safety profile. Together, these findings position 7MW4911 as a promising ADC candidate capable of enhancing therapeutic outcomes in GI cancers.

Claudins are a family of tight junction proteins characterized by four transmembrane domains, with critical roles in epithelial cell junction formation. While claudins localize to tight junctions in healthy tissues, their overexpression in solid tumors leads to aberrant exposure outside of these junctions, making them attractive targets for ADC therapies. CLDN1, in particular, is overexpressed in several solid tumors, including head and neck squamous cell carcinoma (HNSCC), lung squamous cell carcinoma (LUSC), and hepatocellular carcinoma (HCC), where its expression correlates with tumor proliferation, invasion, metastasis, and poor prognosis. Here, we developed a novel CLDN1-targeting ADC, MW-C1, that has shown potent anti-tumor activity across various preclinical models, along with an encouraging safety profile in non-human primates (NHPs). MW-C1 was engineered by conjugating monomethyl auristatin E (MMAE) to a CLDN1-targeting monoclonal antibody using site-specific interchain-disulfide conjugation technology and a cleavable linker, resulting in a drug-to-antibody ratio (DAR) of 4. In vitro, the internalization and cytotoxicity of MW-C1 were assessed in SCC4, Cal27, Huh7, and OVCAR3 cell lines, which express varying levels of CLDN1. In vivo efficacy was evaluated in multiple cell-line-derived xenograft (CDX) and patient-derived xenograft (PDX) models. Toxicology and pharmacokinetic studies were conducted in cynomolgus monkeys. CLDN1 was found to be overexpressed in 85.7% of HNSCC, 91.7% of HCC, 58% of LUSC, and 77% of cervical cancer samples. MW-C1 demonstrated robust binding, rapid internalization, and potent cytotoxicity in CLDN1-positive cancer cell lines. In in vivo studies, MW-C1 induced significant and durable tumor regression in both CDX and PDX models, with varying CLDN1 expression levels. Pharmacokinetic studies revealed favorable properties for MW-C1 in both mice and cynomolgus monkeys, which were attributed to its optimized biophysical characteristics and site-specific conjugation. In a non-GLP NHP toxicology study, MW-C1 was well tolerated, with no significant off-target toxicity or adverse events observed. MW-C1 demonstrated potent anti-tumor efficacy and a favorable safety profile, highlighting its potential as a transformative therapeutic candidate for the treatment of CLDN1-overexpressing malignancies. Zhongfei Li, Jun Yang, Peng Fang, Rongrong Hu, Cuicui Guo, Xiaoding Tan, Hai Wu, Xun Gui. MW-C1, a novel CLDN1-targeting ADC demonstrates compelling anti-tumor efficacy and favorable safety profile in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1573.

Structure of linker-payload complexes and conjugation process of 9MW2821.

Human immunoglobulin G2 (IgG2) is a crucial therapeutic monoclonal antibody (mAb). IgG2 possesses a unique short hinge region characterized by four pairs of interheavy chain disulfide bonds, generating multiple disulfide-bonded isomers, including IgG2-A, IgG2-B, and the intermediate IgG2-A/B. Despite their biological relevance, the characterization of isomers has primarily focused on IgG2-A and IgG2-B, with IgG2-A/B overlooked. In this work, using ion-exchange chromatography and mass spectrometry, we purified isomers of recombinant IgG2κ mAb, identified the conversion mechanism of disulfide bonds, and assessed their biological potencies. The potencies of the isomers were found to be IgG2-A > IgG2-A/B > IgG2-B. These differences may correlate with the increased hydrodynamic radius of IgG2-A and IgG2-A/B compared with IgG2-B. Disulfide bond isomers should be categorized as a critical quality attribute for IgG2 mAb due to the significant potency differences. Our work provides a strategy for purifying a particular disulfide isomer of therapeutic IgG2.

Toxicokinetic parameters of 9MW2821 in cynomolgus monkeys.

Binding affinity and kinetics based on Bio Layer Interferometry

Patient information for prostate cancer formalin-fixed paraffin-embedded samples

Analytical results of 9MW2821 and enfortumab vedotin (EV)

Nectin-4 expression levels and in vitro cytotoxicity

Binding specificity based on enzyme-linked immunosorbent assay.

Binding affinity measured by Biolayer interferometry.