Development and Clinical Trial of M701, an Anti-EpCAM × Anti-CD3 Bispecific Antibody: A Targeted Intraperitoneal Therapy for Malignant Ascites Stemming from Advanced Solid Tumors

Development and Clinical Trial of M701, an Anti-EpCAM × Anti-CD3 Bispecific Antibody: A Targeted Intraperitoneal Therapy for Malignant Ascites Stemming from Advanced Solid Tumors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and Clinical Trial of M701, an Anti-EpCAM × Anti-CD3 Bispecific Antibody: A Targeted Intraperitoneal Therapy for Malignant Ascites Stemming from Advanced Solid Tumors Rongrui Liu, Rongbo Lin, Ning Li, Guiling Li, Tao Zhang, Jun Zhao, and 27 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7204041/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Nov, 2025 Read the published version in Experimental Hematology & Oncology → Version 1 posted 12 You are reading this latest preprint version Abstract Background Malignant ascites (MA) is one of the major complications in advanced epithelial cancer patients and is associated with poor prognosis, poor quality of life, and severe symptoms. No efficient medicine is available for treating MA worldwide. Only paracentesis is recommended by the guidelines in most countries, but with limited efficacy and a short control time. Thus, novel treatments are needed to control MA. Methods An anti-EpCAM × anti-CD3 bispecific antibody, M701, was constructed as a T-cell engager to eliminate tumor cells in the peritoneal cavity. A phase 2 study was performed to evaluate the efficacy and safety of the intraperitoneal (IP) infusion of M701 in advanced epithelial tumor patients with moderate-to-large-scale MA. In this study, 84 patients were enrolled, with 43 in the M701 group receiving paracentesis and IP M701 infusion and 41 in the control group receiving paracentesis alone. Results The major endpoint, median puncture-free survival (PuFS), was 75 days in the M701 group and 25 days in the control group, with a significant difference (p = 0.0065). Subgroup analysis indicated that different types of cancer, including gastric, colorectal, and ovarian cancers, all benefited from the M701 infusion. Patients with higher relative lymphocyte counts (≥ 13%) at baseline received better effects. Compared to those in the control group, the overall survival (OS) of patients in the M701 group was longer (mOS 110 days vs 76 days, p = 0.1443, HR = 0.68). The 6-month survival rates were 33.3% and 12.1% in the two groups, respectively. No additional serious adverse events (SAEs) were detected in the M701 group. The most frequent treatment-related adverse events were anemia and low white blood cell count, which were manageable. M701 infusions did not cause a greater risk than paracentesis alone in the control arm, while all patients were administered systemic treatment. Conclusion When treated with M701, patients with MA had significantly longer puncture intervals and trend of longer survival times. The results were encouraging for patients with MA. A phase III clinical trial of M701 aimed at further validation is ongoing. EpCAM CD3 bispecific antibody malignant ascites intraperitoneal therapy puncture-free survival Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 BACKGROUND Malignant Ascites (MA) refers to the accumulation of fluid in the abdominal cavity due to the presence of various malignant tumors and is most commonly associated with gastrointestinal and gynecological cancers [ 1 – 3 ]. The presence of MA indicates that the primary tumor has metastasized either locally or systemically. The build-up of moderate-to-large volumes of fluid often compresses nearby organs, impairing their function. It can also lead to symptoms such as abdominal pain, breathing difficulty, electrolyte imbalances, hypoproteinaemia, and secondary infections [ 3 , 4 ]. The prognosis is dire, and the disease imposes a heavy burden on patients, significantly worsening their quality of life [ 3 – 5 ]. No globally approved drug is available that specifically targets the treatment of MA, and no established guidelines or consensus on its diagnosis and management are available [ 1 , 4 , 5 ]. Clinically, the primary method of symptom relief involves paracentesis or catheter drainage, which offers immediate alleviation. These procedures are often supplemented by treatments, such as diuretics, or the IP administration of chemotherapy or antiangiogenic agents, based on clinical experience. However, these approaches lack robust evidence from clinical trials and have limitations regarding safety and efficacy [ 5 – 7 ]. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that functions as an adhesion molecule [ 8 , 9 ]. The 39–42 kDa protein consists of a large extracellular domain with two epidermal growth factor-like repeats, a single transmembrane region, and a short cytoplasmic tail of 26 amino acids [ 10 ]. It is extensively expressed in normal epithelial tissues, including gastric, colon, pancreas, ovarian, and lung tissues, and has low expression levels [ 11 , 12 ]. It is frequently overexpressed in various epithelial malignancies and is associated with poor prognosis [ 13 – 17 ]. Treatment of head and neck cancer cells, as well as breast cancer cells, with EpCAM-specific antisense oligonucleotides or siRNAs has led to significant reductions, or even complete inhibition, of cell proliferation, migration, and invasion [ 18 , 19 ]. Moreover, researchers have found EpCAM-positive tumor cells in peritoneal effusions from multiple types of epithelial cancers [ 12 ]. Therefore, EpCAM is a promising target for treating MA [ 14 – 17 ]. Studies have shown that BsAbs can act in either a combinatorial or an obligate manner, with the latter meaning that the same mechanism of action cannot be achieved by simply combining antibodies [ 20 , 21 ]. Over the past two decades, T-cell engagers (TCEs) that specifically bind to a tumor surface antigen and the CD3ε chain of the T-cell receptor have dominated this class of bispecific antibodies; several hundred TCEs have been described, with more than 100 advancing to clinical development[ 22 , 23 ]. A major challenge in the clinical development of TCEs involves the occurrence of cytokine release syndrome, which is largely caused by the activation of on-target T-cells [ 24 , 25 ]. Although this can often be managed via pretreatment using steroids and by step-up dosing, recent efforts have focused on developing CD3ε antibodies with a decrease in CD3ε affinity to uncouple T-cell killing from cytokine secretion [ 26 – 32 ]. Catumaxomab is a bispecific mouse-rat chimeric monoclonal antibody that targets EpCAM and human cluster of differentiation 3 (CD3). It was approved by the EMA in 2009 for treating MA because of its favorable efficacy in reducing the frequency of puncture or drainage [ 33 ]. However, due to commercial reasons, it was withdrawn from the European market in 2017 and reapproved by European Commission in 2025 ( https://www.ema.europa.eu/ ). This EpCAM×CD3 bispecific antibody facilitates the binding of immune cells to tumor cells, boosting the targeted immune response and increasing immune cytotoxicity against tumor cells. Moreover, catumaxomab also binds to CD3 on T cells, triggering their activation and proliferation, causing tumor-killing molecules such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), perforin, and granzyme B to be released [ 33 , 34 ]. Additionally, catumaxomab exhibits antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity against tumor cells [ 33 , 34 ]. The novel human-mouse chimeric monoclonal antibody M701 targets the same antigens as catumaxomab but features a restructured design and robust CMC properties via the CHO cell expression system. With optimized EpCAM and CD3 affinities, M701 is designed for treating MA and malignant pleural effusions caused by solid epithelial tumors. This bispecific antibody has shown significant inhibitory effects on tumor cells derived from MA in preclinical studies, indicating that it is a targeted therapeutic approach [ 35 ]. A Phase I clinical study was performed on epithelial tumor patients with MA, and a Phase Ib study was performed on lung cancer patients with malignant pleural effusion. Both studies showed a favorable safety profile and promising efficacy in controlling MA and malignant pleural effusion [ 36 , 37 ]. Recently, a Phase II clinical trial was performed to further evaluate the safety and efficacy of IP administration of M701 in patients with advanced epithelial solid tumors complicated by MA. METHODS Anti-EpCAM and anti-CD3 BsAb design Anti-EpCAM and anti- CD3 BsAb (M701) is a Fab-scFv-Fc bispecific antibody (bsAb), the Fab moiety is anti-EpCAM, and the scFv moiety is anti-CD3. The anti-EPCAM monovalent unit and the anti-CD3 single-chain unit of M701 were from the anti-EpCAM variable regions of AMG110 (Amgen) and L2K, respectively. The mutations in the CH3 domains of the human IgG1 Fc fragment included T366W-Y407A (knobs-into-holes pair), L368R-K409D (ionic bond “salt bridge”), and D399K-K392D (second salt bridge). The expression system used was CHO-S (Gibco), in which M701 was stably expressed. The purification process was similar to that used to purify classical monoclonal antibodies, including depth filtration, affinity chromatography, low pH, ion-exchange chromatography, nanofiltration, and ultrafiltration/diafiltration (UF/DF). Molecular weight determination by LC-MS Reverse-phase HPLC was performed using an ultrahigh-performance system (UPLC Vanquish, Thermo) that was coupled to an orbitrap (QE) mass spectrometer (Q Exactive Plus, Thermo). Cell binding assay The HCT116 cells were incubated with serially diluted M701 or HCT116 for 1 h at room temperature and then incubated with PE-conjugated anti-human IgG Fc (secondary antibodies) for 30 min in the dark. The binding activity was evaluated by flow cytometry analysis. The same method was used to measure the affinity of M701 and L2K for human T cells. The dissociation constant Kd was calculated using the software GraphPad Prism. To measure the M701-mediated cell bridging, EpCAM-positive NCI-N87 cells were labeled with 2.5 µM CFSE (Invitrogen), and CD3-positive cytokine-induced killer cells (CIKs) were labeled with 0.2 µM PKH26 (Sigma). The cells were washed three times and mixed at a ratio of 1:1. The mixtures were incubated with serially diluted antibodies (0 to 10 µg/mL) for 30 min at 37°C in a 96-well round bottom plate (Corning). M701-mediated cell bridging was evaluated by conducting flow cytometry analysis and is presented as the percentage of cells in the upper right quadrant of an FL1 vs. FL2 scatter plot, which represents the CFSE-PKH26-double-positive population. In vitro cytotoxicity assay CIKs were utilized as effector cells to evaluate the in vitro efficacy of M701. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using Ficoll-Hypaque (Sigma) density gradient centrifugation, following the manufacturer’s protocol. The isolated PBMCs were then co-cultured with anti-CD3 monoclonal antibody, IFN-γ, IL-2, and IL-1α for 14 days to generate CIKs. The bioactivity of M701 was assessed using a fluorescence-activated cell sorting (FACS)-based cytotoxicity assay. Target cells included EpCAM-high cancer cell lines (HCT116, colorectal carcinoma; OVCAR-3, ovarian adenocarcinoma; KATO-III, gastric carcinoma), EpCAM-low cancer cell lines (SK-OV-3, ovarian adenocarcinoma; MDA-MB-231, breast adenocarcinoma), and the EpCAM-negative cancer cell line U87 (glioblastoma). In the assay, 2.0 × 10⁴ CFSE-labeled target cells were co-cultured with CIK effector cells at an effector-to-target (E:T) ratio of 5:1 in 96-well flat-bottom plates (Corning) with serial dilutions of M701 or control antibodies for 24 hours at 37°C in a 5% CO₂ incubator. After incubation, cells were harvested and stained with propidium iodide (PI, Sigma) to assess apoptotic/necrotic populations by flow cytometry. Animal studies Mouse Strains and Housing: Female NOD/SCID and C57BL/6 mice (7–8 weeks old) were purchased from Beijing HFK Bioscience Co., Ltd. Xenograft subcutaneous tumor model: A total of 1.0 × 10⁷ HCT116 cells and 1.0 × 10⁷ CIK cells were mixed and inoculated subcutaneously into the right dorsal flank of 30 NOD/SCID mice. Within 2 hours of inoculation, the mice were randomly assigned to three experimental groups and three control groups (n = 5 per group). Mice in the experimental groups received M701 (0.1, 0.5, or 2.5 mg/kg) via intravenous bolus injection into the lateral tail vein. Mice in the control groups were treated with anti-EpCAM monoclonal antibody (mAb) (2.5 mg/kg), anti-CD3 isotype (2.5 mg/kg), or saline. Treatments were repeated on days 2 and 4. Tumor volumes were measured every three days using a digital caliper, and the tumor volume (mm³) was calculated using the formula: ½ × (length × width²). Syngeneic ascites tumor model: The CT26-mEpCAM cell line, a CT26-derived cell line transfected to express high levels of murine EpCAM, was used in this model. M708, a surrogate of M701, binds both murine EpCAM and murine CD3. The CT26-mEpCAM murine model was established by subcutaneously inoculating 3.0 × 10⁶ CT26-mEpCAM cells into the right dorsal flank of C57BL/6 mice. Starting on day 0 and continuing on days 2, 4, 7, 9, and 11, mice received IP bolus injections of 0.025 mg/kg M708, 0.25 mg/kg M708, 2.5 mg/kg M708, 0.25 mg/kg anti-mEpCAM mAb, 0.25 mg/kg anti-mCD3 isotype, a combination of 0.25 mg/kg anti-EpCAM and 0.25 mg/kg anti-mCD3 isotype, or vehicle (n = 10 per group). Mice were euthanized when the tumor volume reached 2000 mm³ or at the conclusion of the study. Phase II trial design We conducted an open-label, multicenter, randomized, and controlled phase II trial, M70102, in 34 hospitals in China between December 2022 and July 2024 (ClinicalTrials.gov identifier: NCT06266091). The study was conducted following the guidelines of the Declaration of Helsinki and approved by the institutional review board of all involved hospitals. All patients provided signed informed consent before joining any study-related procedure. Trial population Patients aged 18–75 years who were diagnosed with gastrointestinal and ovarian cancers with moderate-to-large-volume symptomatic MA were enrolled. Gastric and colorectal cancer patients had failed at least two lines of systemic treatment, and ovarian cancer patients were platinum-resistant. Additionally, only patients with an Eastern Cooperative Oncology Group (ECOG) performance status of 0–2, life expectancy ≥ 8 weeks and adequate bone marrow function, defined as absolute neutrophil count ≥ 1.5 × 10 9 /L, platelet count ≥ 80 × 10 9 /L, hemoglobin ≥ 9.5 g/dL, serum albumin ≥ 28 g/L, and relative lymphocyte count (RLC) ≥ 10% in peripheral blood, were allowed to participate. Patients whose serum creatinine level was > 1.5 times the upper limit of normal were excluded. A washout period of two weeks before cycle 1, day 1 was mandatory if the patients were administered any IP chemotherapy. Trial intervention The patients were randomly assigned to two groups at a ratio of 1:1. In the M701 group; patients received paracentesis and IP infusions of 50, 400, 400, and 400 µg of M701 on days 1, 4, 11, and 18, respectively. Additional M701 IP infusions could be administered every two weeks without requiring punctures or drainage until the MA was intolerant or patients died. In the control group, patients received paracentesis alone as needed from days 1 to 18. Both arms received systemic tumor treatment as determined by investigators following the guidelines. After day 18, no puncture or drainage was allowed for the patients in either arm until the MA was intolerant. The patients in the control group were allowed to transfer to the M701 group and receive the M701 IP infusion after they reached the major endpoint. Endpoints and statistical analysis The primary endpoint was PuFS, defined as the time to the first repuncture or drainage due to intolerance of MA or death after the last drainage from days 1 to 18, whichever occurred first. The criteria for ascites intolerance include: 1) The patient complains that he/she cannot tolerate the ascites; 2) The presence of a large amount of ascites in the patient is determined by B-ultrasound (the maximum depth of ascites ≥ 4.5 cm); 3) The investigator determines that the patient's total score for ascites symptoms and signs exceeds 7 points (by Likert 4-points scale, see Appendix 1). The secondary efficacy endpoints included OS, progression-free survival (PFS) for the target lesion according to the RECIST 1.1 criteria, and the best objective response rate of MA following the WHO criteria. Safety was assessed based on adverse events reported during the study. All adverse events were coded according to Medical Dictionary for Regulatory Activities (MedDRA 27.0) and graded according to the Common Terminology Criteria for Adverse Events (CTCAE) V5.0. Cytokine release syndrome (CRS) is the only listed adverse event of special interest (AESI). The sample size was determined by conducting log-rank tests. The assumption was that the median PuFS of the control group was 18 days, and the hazard ratio (HR) between the experimental group and the control group was 0.5. With a power of 80%, alpha = 5%, and a dropout rate of 10%, 60–80 patients were needed. Fisher’s exact test was conducted to analyze nominal variables, and the Mann-Whitney U test was conducted to analyze continuous variables. The survival curves were estimated using the Kaplan-Meier method. All data were analyzed using Statistical Analysis System software (SAS® 9.4 or higher version). To conduct cell and animal studies, statistical analysis was performed using Prism 6 (GraphPad Software Inc., La Jolla, CA). The differences between groups were determined by conducting two-sided Student’s t-tests. All data were presented as the mean ± SD. All results were considered to be statistically significant at P < 0.05. Pharmacokinetics and immunogenicity Blood and ascites samples were collected to evaluate the pharmacokinetics and immunogenicity of M701. The pharmacokinetic analysis set (PKAS) and immunogenicity analysis set (IS) included all patients who received at least one dose of M701 infusion and from whom adequate drug concentration or anti-drug antibody (ADA) measurements were obtainable during the trial. Blood samples for pharmacokinetic and immunogenicity analysis were collected on days 1, 4, 18, 46, and 88 and every two treatment cycles 1 h before M701 infusion. Ascites samples for PK and immunogenicity analysis were collected on days 1, 18, 32, 46, and 88 and every two treatment cycles 24 h before M701 infusion. The serum and ascites concentrations of M701 or ADA were determined using validated methods. RESULTS Characteristics of M701 M701 consists of two distinct units, including a monovalent unit comprising a heavy chain/light chain pair and a single-chain unit (Fig. 1 a). In the single-chain unit, the single-chain variable fragment (scFv) is constructed in a variable heavy chain (VH)-variable light chain (VL) orientation, with VH and VL connected in-frame by a (Gly 4 Ser) 3 linker, and the scFv is fused to the human IgG1 Fc fragment to form the single-chain This design prevents mismatched pairing and increases the manufacturability of the antibody. The glycosylated and deglycosylated intact mass of M701 was 128.5kDa and 125.6kDa. Under reducing conditions, the molecular weight (Mw) of the light chain was 24.1 kDa, the glycosylated Mw of the heavy chain was about 50.8 kDa and the single-chain was 53.6 kDa, the deglycosylated Mw of the heavy chain was about 49.4 kDa, and the single-chain was 52.1 kDa (Fig. 1 a). The mutations in the Fc fragments of M701 were based on a salt bridge and knobs-into-holes (KIHs). The specific modifications were T366W, K392D, and K409D on the Fc of monovalent unit and L368R, D399K, and Y407A on the Fc of single-chain unit. M701 exhibited moderate affinity and dose-dependent cell bridging The cell binding activity of M701 was evaluated by conducting flow cytometry assays. The results indicated that the affinity of M701 (Kd = 32 nM) was about two times weaker than that of the anti-EpCAM mAb (Kd = 16 nM) for HCT116 cells (Fig. 1 b) and significantly weaker than that of L2K for human T cells (M701 Kd = 33.7 nM, L2K Kd = 0.6 nM) (Fig. 1 c). Additionally, the mechanism of M701-mediated recruitment of CD3-positive cells to EpCAM-positive cells was investigated. CIKs expressing CD3 were labeled with PHK26, and EpCAM-positive NCI-N87 cells were labeled with CFSE. The proportion of double-positive cells was less than 10% in the absence of M701 (Fig. 1 d). In contrast, in the presence of 10 µg/mL M701, up to 40% of the total cell population was double-positive for M701-mediated cell bridging. The enhanced level of cell bridging mediated by M701 was also dose-dependent. Neither the anti-EpCAM mAb nor L2K could mediate the cell bridging at any concentration tested. M701 potently redirected lysis to EpCAM-positive tumor cells We evaluated the cytotoxic effects of M701, mediated by CIKs, on cancer cells with varying levels of EpCAM expression. Target cells with different EpCAM expression levels (Fig. 2 a) were incubated with increasing concentrations of M701 and CIKs at an E:T ratio of 5:1. M701 showed potent cytotoxicity against HCT116, NCI-N87, and KATO III cells (Fig. 2 b), all of which express high levels of EpCAM. However, M701 displayed minimal cytotoxic effects on SK-OV-3 and MDA-MB-231 cells, which express very low levels of EpCAM (Fig. 2 b). Additionally, M701 did not induce cytotoxicity in EpCAM-negative U87 cells, confirming the specificity of the M701-mediated cell lysis (Fig. 2 b). M701 specifically induces potent cytotoxicity in EpCAM-positive tumor cells, with minimal effects on EpCAM-negative cells, highlighting its potential as a targeted therapeutic agent for EpCAM-expressing cancers. M701 and surrogate M708 exhibited potent antitumor efficacy in solid and ascites tumor models In a xenograft subcutaneous tumor model, M701 demonstrated superior efficacy in inhibiting the growth of human colon cancer cells (HCT116) in NOD-SCID mice compared to control antibodies (Fig. 2 c). As a surrogate of M701, M708 possesses the same Fab-scFv-Fc structure and binds both murine EpCAM and murine CD3. In a syngeneic ascites tumor model, IP injection of M708 significantly prolonged the survival of mice with malignant ascites at low, medium, and high doses (Fig. 2 d). These indicates that a bsAb targeting EpCAM and CD3 has a strong inhibitory effect on EpCAM-positive tumors and the malignant ascites caused by these tumors. Baseline characteristics of patients in the Phase II trial From November 2021 to July 2024, 84 patients were enrolled, 43 patients were assigned to the M701 group, 41 patients were assigned to the control group, and one patient in the control group withdrew from the study after initial screening. In total, 83 patients who were administered at least one M701 infusion in the M701 group or one therapeutic paracentesis in the control group were included in the full analysis set (FAS). According to the protocol, seven patients in the control group were transferred to the M701 group to receive M701 via IP infusion. The CONSORT flow diagram are shown in Fig. 3 . The baseline characteristics of the intent-to-treat (ITT) population are shown in Table 1 , and the patients and disease characteristics were balanced between the M701 group and the control group. Most patients in both arms had gastric cancer, accounting for 48.8% of those in the M701 group and 45.0% of those in the control group. The percentage of patients with prior paracentesis was 65.1% in the M701 group and 65.0% in the control group, and the median number of patients with prior paracentesis was one and two in the M701 and control groups, respectively. The median number of prior lines of systemic treatment in both groups was two. A total of 30.2% and 32.5% of patients in the M701 and control groups, respectively, received more than three lines of systemic treatment. Table 1 Patient characteristics in ITT (N = 84) Characteristic ITT Set M701 (N = 43) Control (N = 41) Age(years) Mean (SD) 54.8 (8.99) 54.4 (10.64) Median (P25,P75) 54 (50.0–59.0) 54 (49.0-62.5) ≥ 60 10 (23%) 14 (34%) Gender Male 14 (33%) 14 (34%) Female 29 (67%) 27 (66%) ECOG 0 4 (9%) 2 (5%) 1 34 (80%) 34 (83%) 2 4 (9%) 5 (12%) Cancer Type Gastric 21 (49%) 19 (46%) Ovarian 13 (30%) 13 (32%) Colorectal 8 (19%) 8 (20%) Fallopian tube cancer 1 (2%) 0 Primary peritoneal cancer, 0 1 (2%) If maintain the system treatment regimen after enrolled Yes 3 (7%) 3 (7%) No 40 (93%) 38 (93%) Clinical Stages II 1 (2%) 1 (2%) III 10 (23%) 3 (7%) IV 30 (70%) 37 (90%) Unknown 2 (5%) 0 Previous treatment IP treatments 24 (56%) 21 (51%) Paracentesis 27 (63%) 25 (61%) Previous paracentesis frequency (%) 0 37.2% 46.3% 1–3 51.2% 31.7% ≥ 4 times 11.6% 17.1% Unknown 0 4.9% Treatment outcomes To evaluate the efficacy of M701 in controlling MA, PuFS was set as the primary endpoint and directly reflected the ability of treatment to prevent the reaccumulation of ascites in the peritoneal cavity. Median PuFS was significantly longer in the M701 group in the ITT population (75 vs. 25 days) (Fig. 4 a), with an HR of 0.43 (95% CI: 0.22, 0.81) and p = 0.0065. The one-month PuFS rates were 65.2% in the M701 group and 40.6% in the control group, whereas the two-month PuFS rates were 50.3% in the M701 group and only 22.1% in the control group (Table 2 ). A significant difference in the two-month PuFS rates was found between the two groups (p = 0.001), indicating that compared to the control group, the M701 group had a significantly longer time without the need for peritoneal drainage. Table 2 PuFS in the FAS and RLC ≥ 13% of populations FAS population RLC ≥ 13% population M701 Control M701 Control N 43 40 31 36 Median PuFS (95%CI), Day a 75 (29–153) 25 (12–32) 125 (45–174) 22 (11–32) P value (Log-rank test) b 0.0065 0.0003 Hazard Ratio(95%CI) c 0.43 (0.22, 0.81) 0.23(0.10, 0.55) Half a month PuFS rate (95%CI) a 80.8 (65.2, 89.9) 67.2 (49.5, 79.9) 86.6 (68.1, 94.8) 63.1 (44.3, 77.1) 1-month PuFS rate (95%CI) a 65.2 (47.1, 78.4) 40.6 (23.6, 56.9) 73.4 (51.5, 86.6) 38.2 (21.1, 55.2) 2-month PuFS rate (95%CI) a 50.3 (31.8, 66.3) 22.1 (8.2, 40.2) 67.8 (44.7, 82.9) 18.3 (5.5, 37.0) a. Based on the Kaplan-Meier estimates; b. Based on the Log-rank test; c.Based on the Cox regression. The PuFS in different subpopulations was also analyzed. The different subpopulations benefited from M701 treatment, regardless of sex, age, type of cancer, clinical stage, previous treatment history, and time point of disease diagnosis. Compared to the women in the control group, women with Stage IV disease who previously received local chemotherapy or paracentesis significantly benefited from M701 treatment (Fig. 5 ). An in vitro study revealed that M701 could bridge T lymphocytes to EpCAM-positive tumor cells, which activated T lymphocytes to kill tumor cells. Therefore, the rationale that patients with high T lymphocyte counts respond better to M701 treatment was verified in the subpopulation with a high RLC in whole blood cells. In the population with RLC ≥ 13% at baseline, the M701-treated patients showed an even greater PuFS benefit than individuals in the control group (Fig. 4 b). The median PuFS times were 125 days and 22 days, respectively (p = 0.0003, HR = 0.23 (0.10, 0.55)) (Table 2 ). Tumor response to systemic therapy was not found in either group (Objective response rate = 0%). The PFS in those two groups was not significantly different (HR = 0.80 and p = 0.40) (Fig. 6 c). The final analysis of survival suggested that treatment with M701 provided a trend of survival benefit with HR = 0.68 and p = 0.1443 (Fig. 6 a). The six-month survival rate in the M701 group was considerably higher than that in the control group (33.3% vs 12.1%) (Table 3 ). In the population with RLC ≥ 13% at baseline, the M701-treated patients received significant benefits in overall survival (HR = 0.46 and p = 0.0155) (Fig. 6 b) Table 3 Overall survival in the FAS and RLC ≥ 13% of populations FAS population d RLC ≥ 13% population d M701 Control M701 Control N 43 33 31 29 Median OS(95%CI), Daya 110 (82, 177) 76 (55, 116) 152 (94, 244) 76 (55, 116) P value(Log-rank test)b 0.1443 0.0155 Strata Hazard Ratio (95%CI)c 0.68 (0.40, 1.15) 0.46 (0.24, 0.88) 2-month survival rate(95%CI)a 75.9 (59.9, 86.3) 66.7 (47.9, 80.0) 86.5 (67.9, 94.7) 69.0 (48.8, 82.5) 3-month survival rate(95%CI)a 58.8 (42.3, 72.0) 45.5 (28.2, 61.2) 72.7 (52.7, 85.3) 44.8 (26.5, 61.6) 6-month survival rate(95%CI)a 33.3 (19.4, 47.8) 12.1 (3.8, 25.5) 40.1 (22.3, 57.3) 13.8 (4.3, 28.6) a. Based on the Kaplan-Meier estimates; b. Based on the Log-rank test; c. Based on the Cox regression; d. The transferred patients in the control group were excluded. M701 exposure and Safety profiles Among the patients in the M701 group and the control group transferred to the M701 group, 50 patients received between 1 and 16 infusions of M701 individually. The median number of infusions was four, with an average of five infusions per patient. All patients in the control group received a puncture and an indwelling catheter. Among them, 85% carried two or more drainages from days 1 to 18. Although the IP infusion of M701 is a kind of local therapy, it is hypothesized that it will increase the risk to patients because of T lymphocyte activation. In this study, 50 subjects received at least one dose of M701 and systemic therapy, while 40 subjects in the control group received systemic therapy and paracentesis. Treatment emergent adverse events (TEAEs) occurred in 47 subjects (94.0%) in the experimental group and 37 subjects (92.5%) in the control group, with similar TEAE incidence rates between the two groups. The incidence of ≥ Grade 3 TEAE occurred in 52.0% and 55.0% of the subjects in the two groups, respectively. The results showed that 50% of subjects in both groups encountered SAEs. The details of the safety profile are summarized in Table 4 . Compared to paracentesis alone, the local infusion of M701 combined with systemic therapy did not cause risk to patients. Table 4 Overall adverse events in SS M701 (N = 50) Control (N = 40) Case (incidence %) Case (incidence %) TEAE 47(94.0) 37(92.5) M701 related AE 40 (80.0) N/A Systemic therapy related AE 36 (72.0) 30 (75.0) ≥ Grade 3 TEAE 26 (52.0) 22(55.0) ≥ Grade 3 M701 related AE 24 (48.0) N/A SAE 25(50.0) 20(50.0) CRS 2 (4.0) 0 (0) In the M701 group, the most common SAEs included gastrointestinal disorders (16.0%), laboratory examinations (14.0%), blood and lymphatic system disorders (12.0%), infections and infestations (10.0%), general disorders and administration site conditions (6.0%), and disorders related to metabolism and nutrition (6.0%). In the control group, the most common SAEs included gastrointestinal disorders (22.5%), infections and infestations (15.0%), disorders related to metabolism and nutrition (10.0%), and general disorders and administration site conditions (4.0%). The details of the SAEs are summarized in Table 5 . Table 5 List of patient SAEs in the safety set (≥ 5%) M701 group (N = 50) Control group (N = 40) Total (N = 90) By SOC Cases/Incidence(%) Cases/Incidence(%) Cases/Incidence(%) Patients with ≥ 1 SAE event 25 (50.0) 20 (50.0 ) 45 (50.0 ) Gastrointestinal disorders 8 (16.0) 9 (22.5) 17 (18.8) Intestinal obstruction 3(6.0) 1(2.5) 4(4.4) Lab examinations 7(14.0) 1(2.5) 8(8.9) White blood cell count decreased 3(6.0) 0 3(3.3) Platelet count decreased 3(6.0) 0 3(3.3) Neutrophil count decreased 3(6.0) 0 3(3.3) Blood and lymphatic system disorders 6 (12.0) 0 6 (6.7) Anemia 5 (10.0) 0 5 (5.5) Infections and infestations 5 (10.0) 6 (15.0) 11 (12.2) General disorders and administration site conditions 3(6.0) 2 (5.0) 5 (5.6) Metabolism and nutrition disorders 3(6.0) 4(10.0) 7 (7.8) Decreased appetite 2(4.0) 3(7.5) 5(5.6) Compared to the individuals in the control group, the patients in the M701 group had a greater incidence of SAE in terms of decreased blood cells and anemia, which may be related to the activation of T-lymphocytes. However, those SAEs in blood cells were manageable and reversible. No patients discontinued the M701 treatment because of severe adverse reactions. The ASEI in this study was CRS which were frequently reported in TCEs treated patients. CRS occurred in 4% of the M701 group and the CTCAE grades ranged from 1 to 2. Pharmacokinetics and immunogenicity Among the 50 patients who received at least one infusion of M701, 42 had at least one blood or ascites sample collected for pharmacokinetics and immunogenicity analysis. These patients were included in the PK analysis set and immunogenicity set. The M701 concentration was below the lower limit of quantitation in most of the serum and ascites samples. The highest serum concentration of M701 was found on day 18, and the arithmetic mean was 6.53 ng/mL. The highest concentration of M701 in ascites was found on day 18, and the arithmetic mean was 26.5 ng/mL. In the serum, the rate of ADA positivity was 64.3%, which was caused by the M701 treatment, whereas in the ascites, it was 45.2%. As the number of administrations increased, the titer of ADA increased, both in the serum and ascites. However, the ADA titer in ascites was considerably lower than that in serum, and the ratio was about 0.1–0.3. DISCUSSION MA has been a great challenge for cancer patients and oncologists for decades. Although new drugs for systemic cancer treatment have emerged in the last two decades, significantly prolonging the survival of cancer patients, the issue of MA has become more prominent for patients with advanced-stage cancer. Different drugs or devices have been tested in many clinical trials, including TNFα, IL-2, chemotherapy, filtered ascites, and catumaxomab, among which only catumaxomab completed a controlled randomized clinical trial and was approved by the EMA for MA treatment in 2009[ 2 ]. Due to certain commercial reasons, Catumaxomab was withdrawn from the European market in 2017. However, after eight years, no new, more effective treatments have emerged on the market. As a result, in 2025, the European Commission re-approved the marketing of catumaxomab in the European Union for the treatment of malignant ascites, based on previous clinical research results. In this study, M701 was constructed on an asymmetric bispecific antibody platform (YBODY®) developed by YZYBIO, Inc., with favorable characteristics concerning pairing efficacy, yield, and stability using the CHO cell expression system. In vitro assays revealed that M701 specifically trafficked CD3-positive cells to EpCAM-positive cells and caused lysis of only tumor cells with moderate-to-high EpCAM expression, indicating that M701 has few side effects on normal tissue. In xenograft tumor-bearing mice, M701 inhibited tumor growth but neither the anti-EpCAM antibody nor the anti-CD3 antibody performed such inhibition. In an immunocompetent C57BL/6 mouse model, a surrogate molecule targeting murine EpCAM and CD3 also prolonged the survival of mice with MA. To evaluate the safety and efficacy of M701, this phase II trial was conducted on advanced cancer patients with moderate-to-large-volume symptomatic MA who had failed at least two lines of systemic treatment or were platinum-resistant. The patients in the M701 group received an IP infusion of M701 after drainage, whereas the patients in the control group received only drainage. Moreover, both groups received the systemic treatments selected by the investigator based on the Chinese clinical guidelines. The results of the safety evaluation revealed that these combinations were well-tolerated and that local infusion of M701 did not increase the risk to patients. The highest incidence of M701-related SAEs was the abnormal level of all kinds of blood cells, including decreased white blood cells, platelets, and anemia, which may be due to the activation of T lymphocytes. However, those AEs were transient and reversible. No patient in the M701 group discontinued treatment because of abnormal laboratory examinations. Owing to its asymmetric structure and human-mouse chimerism, M701 has a moderate CD3 affinity and low immunogenicity, which indicates that M701 has the innate characteristics of low incidence of CRS and human anti-mouse antibody (HAMA) reaction. Therefore, the IP infusion of M701 was performed more than 10 times without causing strong CRS or HAMA. Additionally, hepatic toxicity, which is a common ADR caused by catumaxomab, was not observed in M701-treated patients [ 38 – 40 ]. Moreover, repeated M701 IP infusions did not cause prominent abdominal compartment separation, which is frequently reported in patients who have undergone IP chemotherapy [ 41 – 42 ]. Regarding effectiveness in controlling MA, the M701 group showed an encouraging effect, with a median PuFS of 75 days, which was better than that of the control group (25 days) and the historical data of a pivotal trial of catumaxomab (46 days) [ 39 ]. The extra benefit may come from systemic treatment since the control group in this study had a longer PuFS than the historical data of the control group in the catumaxomab pivotal study (11 days) [ 39 ]or the lower grade and incidence of CRS and relative adverse drug reactions. The patients in the control group in this study had more drainage than those in the catumaxomab pivotal study (only once); this fact may also have contributed to the prolonged PuFS in the control group. Systemic chemotherapy or antiangiogenic drugs may also weaken hematopoietic function, which may decrease the effect of T-cell-engaging antibodies and increase the risk of M701 infusion. Therefore, we could only conclude that M701 infusion significantly prolonged the puncture/drainage interval with regular systemic treatments. According to the analysis of PuFS in the subpopulation, some subpopulations, including female patients and patients who previously underwent chemotherapy or paracentesis, received significant benefits. As a local treatment, M701 infusion is unlikely to improve OS because of the low dose of M701 (400 µg) and limited distribution in peripheral blood. No objective response was found during this phase II study in either the M701 or the control group (data not shown). However, the M701 group showed trends of longer OS in the FAS population and most subpopulations. Gastrointestinal cancer patients have longer OS than ovarian cancer patients, which is consistent with the results of pivotal trials of catumaxomab in MA patients. The increase in OS may be due to improvements in the physical status or willingness to continue systemic treatment of patients with a heavy burden of MA. Since M701 kills tumor cells in the peritoneal cavity by recruiting and activating T lymphocytes, we hypothesized that patients with higher lymphocyte counts at baseline may receive greater benefits from M701 treatment. The results of the study revealed that patients with ≥ 13% RLC in peripheral blood had better PuFS than the FAS population (125 days vs. 75 days). These patients also showed significantly longer OS (152 days vs 76 days). Over 40% of patients in this subgroup survived for six months, whereas most MA patients were found to survive for only 2–3 months [ 2 , 4 ]. This trend was also observed in the pivotal trial of catumaxomab as well [ 43 ]. In the phase II study of M701 in MA patients, the participants in both groups received the systemic treatment recommended by the Chinese clinical guidelines for gastrointestinal cancer patients who had failed two lines of chemotherapy and for ovarian cancer patients who were resistant to platinum-based chemotherapy. Systemic treatments may affect PuFS and OS, however, considering that the allocation of tumor types and the number of previous treatment lines in the two groups were balanced, the bias caused by systemic therapies should be minimal. Moreover, there are still certain limitations in this study, including: 1) the sample size is relatively small, and the degree of benefit for patients with different types of tumors has not yet been determined; 2) the QoL data at different time points were not effectively collected. Therefore, further validation of the clinical value of M701 is needed in larger and more well-designed clinical studies. Given its promising efficacy and safety profile, multiple-center pivotal stage III clinical research is ongoing to test the efficacy and safety of M701 in China. Further clinical studies are planned with patients outside China. The importance and benefits of repeated infusion of M701 or rechallenge with M701 after MA intolerance require further evaluation in those clinical trials. Abbreviations MA Malignant Ascites PuFS Puncture-free survival OS Overall Survival SAE Serious adverse event TEAE Treatment emergent adverse event AESI Adverse event of special interest EpCAM Epithelial cell adhesion molecule TCEs T-cell engagers CD3 Cluster of differentiation 3 CRS Cytokine release syndrome TNF- Tumor necrosis factor-alpha IFN- Interferon-gamma bsAb bispecific antibody CIKs Cytokine-induced killer cells ECOG Eastern Cooperative Oncology Group RLC Relative lymphocyte count IP Intraperitoneal PFS Progression-free Survival CTCAE Common Terminology Criteria for Adverse Events HR Hazard ratio ADA Anti-drug antibody Mw Molecular weight KIH Knobs-into-hole FAS Full analysis set HAMA Human anti-mouse antibody Declarations Acknowledgements We would like to thank all subjects and their families who participated in this study and all investigators and research personnel in those study centers. Authors' contributions Conception and design: J.X. and P.Z.; development of methodology: R.L., J.Z., S.H. and J.X; investigation, resources, and data curation: R.L., R.L., N.L., G.L., T.Z., J.Z, J.L., M.S., K.W., H.A., W.Z., H.X., S.Z., M.Z., W.D., Y.B., J.Z., H.T, F.Y., Y.K., Q.X., N.X., Y.D., Q.C., Y.L., H.Y., F.S., Z.X., X.X, and J.X.; analysis and interpretation of data: R.L., J.Z., S.H. and J.X.; writing (review and editing): R.L., J.Z., S.H. and J.X.; study supervision: J.X. and P.Z.. All authors contributed to manuscript editing and approved the submission of the manuscript. Funding This study was completely sponsored by Wuhan YZY Biopharma Co. Ltd. Ethics approval and consent to participate The clinical study was conducted according to the Declaration of Helsinki and was approved by the institutional review board of each hospital. Informed consent was taken from all patients before participating in any study-related procedure. Animal Studies were approved by Huazhong University of Science and Technology Experimental Animal Ethics Committee. Consent for publication This manuscript has not been previously published and is not under consideration for publication elsewhere. Competing interests Dr. Pengfei Zhou, Dr. Shaoyi Huang and Dr. Jing Zhang are the employees of Wuhan YZY Biopharma Co. Ltd, which sponsored this study and owned the commercial development rights of M701. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors’ detail 1 Department of Oncology, The First Medical Center, Chinese PLA General Hospital,28 Fuxing Road, Beijing 100039, China; 2 Department of Abdominal Oncology, Fujian Cancer Hospital, No.420 Fuma Road, Fuzhou 350000, China; 3 Department of Gastrointestinal Oncology, The Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer Hospital, No.127 Dongming Road, Zhengzhou 450000, Henan, China; 4 Department of Gynecologic Oncology, Cancer Center of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefang Avenue, Wuhan 430000, Hubei, China; 5 Department of Abdominal Oncology, Cancer Center of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefang Avenue, Wuhan 430000, China; 6 Department of Oncology, Changzhi People's Hospital, The Affiliated Hospital of Changzhi Medical College, No.502 Changxing Middle Road, Changzhi 046000, China; 7 Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, No.55 Zhenhai Road, Xiamen 361005, China; 8 Department of Oncology, Jinan Central Hospital affiliated to Shandong University, No.105 Jiefang Road, Jinan 250013, China; 9 Department of Gynecological Oncology, Tianjin Medical University Cancer Institute and Hospital, West Huan-Hu Road, Tianjin300060, China; 10 Department of Gastrointestinal Oncology, The Cancer Center, Shanxi Bethune Hospital, Shanxi Medical University, No.99 Longcheng Street,Taiyuan 030032, China; 11 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, No.1 Jianshe Dong Road, Zhengzhou 450052, China; 12 Department of Medical Oncology, Hubei Cancer Hospital, No 116 Zhuodaoquan South Road, Wuhan 430079, China; 13 Department of Chemotherapy Oncology, Xiangya Hospital of Central South University, No.87 Xiangya Road, Changsha 410008 , China; 14 Department of Oncology, The second hospital of Anhui medical university, No.678 Furong Road, Hefei 230601, China; 15 Department of Gynecologic Oncology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, No.17 Qihelou, Beijing 100069, China; 16 Department of Gastrointestinal Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin150040, China; 17 Department of Gastroenterology, Liaoning Cancer Hospital & Institute,No.44 Xiaoheyan Road, Shenyang 110042, China; 18 Department of Gastroenterology, Shandong Cancer Hospital, No.440 Jiyan Road, Jinan 250117, China; 19 Department of Gastroenterology, The Fourth Hospital of Hebei Medical University, No.12 Jiankan Road, Shijiazhuang 050011, China; 20 Department of Gynecological Oncology, Obstetrics and Gynecology Hospital of Fudan University,No.419 Fangxie Road, Shanghai 200011, China; 21 Department of Hepato-Pancreato-Biliary & Gastric Medical Oncology, Zhejiang Cancer Hospital, No. 1 East Banshan Road, Hangzhou 310022, China; 22 Department of Oncology, The First Affiliated Hospital, Zhejiang University School of Medicine,No.79 Qingchun Road, Hangzhou 310003, China; 23 Department of Oncology, The Sixth Affiliated Hospital, Sun Yat-sen University, No. 26 Yuancun Er Heng Road, Guangzhou 510655, China; 24 Department of Obstetrics and Gynecology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, No.107 Yanjiang Road West, Guangzhou 510120, China; 25 The First Department of Chemotherapy, Affiliated Cancer Hospital of Guangxi Medical University, 71 Hedi Road, Nanning 530021, China; 26 Department of Gynaecology, Yunnan Cancer Hospital, No.519 Kunzhou Road, Kunming 650118, China; 27 Department of Oncology, The First Affiliated Hospital of Bengbu Medical University, 287 Changhuai Road, Bengbu, China; 28 Department of Oncology, Nanshi Hospital of Nanyang, No.130 Zhongzhou West Road, Nanyang 473001, China; 29 Department of Oncology, The First Affiliated Hospital of Nanchang University, No.17 Yongwai Main Street, Nanchang 330006, China; 30 Wuhan YZY Biopharma Co. Ltd., C2-1 Building, Guanggu Biolake, No. 666, Gaoxin Avenue, Wuhan 430000, China; 31 Department of Oncology, The Fifth Medical Center, Chinese PLA General Hospital, No.8 Dongdajie, Beijing 100071, China. References Saif MW, Siddiqui IA, Sohail MA. Management of ascites due to gastrointestinal malignancy. Ann Saudi Med. 2009;29(5):369–77. Ayantunde AA, Parsons SL. Pattern and prognostic factors in patients with malignant ascites: a retrospective study. Ann Oncol. 2007;18(5):945–9. Parsons SL, Watson SA, Steele RJ. Malignant ascites. Br J Surg. 1996;83(1):6–14. Sangisetty SL, Miner TJ. Malignant ascites: A review of prognostic factors, pathophysiology and therapeutic measures. World J Gastrointest Surg. 2012;4(4):87–95. Ikegami T, Ishiki H, Kadono T, Ito T, Yokomichi N. Narrative review of malignant ascites: epidemiology, pathophysiology, assessment, and treatment. Ann Palliat Med. 2024;13(4):842–57. Matsusaki K, Aridome K, Emoto S, Kajiyama H, Takagaki N, Takahashi T, Tsubamoto H, Nagao S, Watanabe A, Shimada H, et al. Clinical practice guideline for the treatment of malignant ascites: section summary in Clinical Practice Guideline for peritoneal dissemination (2021). Int J Clin Oncol. 2022;27(1):1–6. Stukan M. Drainage of malignant ascites: patient selection and perspectives. Cancer Manag Res. 2017;9:115–30. Litvinov SV, Velders MP, Bakker HA, Fleuren GJ, Warnaar SO. Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule. J Cell Biol. 1994;125(2):437–46. Balzar M, Briaire-de Bruijn IH, Rees-Bakker HA, Prins FA, Helfrich W, de Leij L, Riethmüller G, Alberti S, Warnaar SO, Fleuren GJ, et al. Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions. Mol Cell Biol. 2001;21(7):2570–80. Balzar M, Winter MJ, de Boer CJ, Litvinov SV. The biology of the 17-1A antigen (Ep-CAM). J Mol Med (Berl). 1999;77(10):699–712. Went P, Vasei M, Bubendorf L, Terracciano L, Tornillo L, Riede U, Kononen J, Simon R, Sauter G, Baeuerle PA. Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers. Br J Cancer. 2006;94(1):128–35. Went PT, Lugli A, Meier S, Bundi M, Mirlacher M, Sauter G, Dirnhofer S. Frequent EpCam protein expression in human carcinomas. Hum Pathol. 2004;35(1):122–8. Yu S, Li A, Liu Q, Yuan X, Xu H, Jiao D, Pestell RG, Han X, Wu K. Recent advances of bispecific antibodies in solid tumors. J Hematol Oncol. 2017;10(1):155. Gastl G, Spizzo G, Obrist P, Dünser M, Mikuz G. Ep-CAM overexpression in breast cancer as a predictor of survival. Lancet. 2000;356(9246):1981–2. Spizzo G, Obrist P, Ensinger C, Theurl I, Dünser M, Ramoni A, Gunsilius E, Eibl G, Mikuz G, Gastl G. Prognostic significance of Ep-CAM AND Her-2/neu overexpression in invasive breast cancer. Int J Cancer. 2002;98(6):883–8. Spizzo G, Went P, Dirnhofer S, Obrist P, Moch H, Baeuerle PA, Mueller-Holzner E, Marth C, Gastl G, Zeimet AG. Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. Gynecol Oncol. 2006;103(2):483–8. Varga M, Obrist P, Schneeberger S, Mühlmann G, Felgel-Farnholz C, Fong D, Zitt M, Brunhuber T, Schäfer G, Gastl G, et al. Overexpression of epithelial cell adhesion molecule antigen in gallbladder carcinoma is an independent marker for poor survival. Clin Cancer Res. 2004;10(9):3131–6. Münz M, Kieu C, Mack B, Schmitt B, Zeidler R, Gires O. The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation. Oncogene. 2004;23(34):5748–58. Osta WA, Chen Y, Mikhitarian K, Mitas M, Salem M, Hannun YA, Cole DJ, Gillanders WE. EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res. 2004;64(16):5818–24. Brinkmann U, Kontermann RE. Bispecific antibodies. Science. 2021;372(6545):916–7. Labrijn AF, Janmaat ML, Reichert JM, Parren P. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019;18(8):585–608. Goebeler ME, Bargou RC. T cell-engaging therapies - BiTEs and beyond. Nat Rev Clin Oncol. 2020;17(7):418–34. van de Donk N, Zweegman S. T-cell-engaging bispecific antibodies in cancer. Lancet. 2023;402(10396):142–58. Leclercq G, Steinhoff N, Haegel H, De Marco D, Bacac M, Klein C. Novel strategies for the mitigation of cytokine release syndrome induced by T cell engaging therapies with a focus on the use of kinase inhibitors. Oncoimmunology. 2022;11(1):2083479. Leclercq-Cohen G, Steinhoff N, Albertí Servera L, Nassiri S, Danilin S, Piccione E, Yángüez E, Hüsser T, Herter S, Schmeing S, et al. Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies. Clin Cancer Res. 2023;29(21):4449–63. Mandikian D, Takahashi N, Lo AA, Li J, Eastham-Anderson J, Slaga D, Ho J, Hristopoulos M, Clark R, Totpal K, et al. Relative Target Affinities of T-Cell-Dependent Bispecific Antibodies Determine Biodistribution in a Solid Tumor Mouse Model. Mol Cancer Ther. 2018;17(4):776–85. Staflin K, Zuch de Zafra CL, Schutt LK, Clark V, Zhong F, Hristopoulos M, Clark R, Li J, Mathieu M, Chen X et al. Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody. JCI Insight 2020, 5(7). Zuch de Zafra CL, Fajardo F, Zhong W, Bernett MJ, Muchhal US, Moore GL, Stevens J, Case R, Pearson JT, Liu S, et al. Targeting Multiple Myeloma with AMG 424, a Novel Anti-CD38/CD3 Bispecific T-cell-recruiting Antibody Optimized for Cytotoxicity and Cytokine Release. Clin Cancer Res. 2019;25(13):3921–33. Trinklein ND, Pham D, Schellenberger U, Buelow B, Boudreau A, Choudhry P, Clarke SC, Dang K, Harris KE, Iyer S, et al. Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies. MAbs. 2019;11(4):639–52. Dang K, Castello G, Clarke SC, Li Y, Balasubramani A, Boudreau A, Davison L, Harris KE, Pham D, Sankaran P et al. Attenuating CD3 affinity in a PSMAxCD3 bispecific antibody enables killing of prostate tumor cells with reduced cytokine release. J Immunother Cancer 2021, 9(6). Liu CY, Ahonen CL, Brown ME, Zhou L, Welin M, Krauland EM, Pejchal R, Widboom PF, Battles MB. Structure-based engineering of a novel CD3ε-targeting antibody for reduced polyreactivity. MAbs. 2023;15(1):2189974. Pouleau B, Estoppey C, Suere P, Nallet E, Laurendon A, Monney T, Pais Ferreira D, Drake A, Carretero-Iglesia L, Macoin J, et al. Preclinical characterization of ISB 1342, a CD38 × CD3 T-cell engager for relapsed/refractory multiple myeloma. Blood. 2023;142(3):260–73. Sebastian M, Kuemmel A, Schmidt M, Schmittel A. Catumaxomab: a bispecific trifunctional antibody. Drugs Today (Barc). 2009;45(8):589–97. Seimetz D, Lindhofer H, Bokemeyer C. Development and approval of the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3) as a targeted cancer immunotherapy. Cancer Treat Rev. 2010;36(6):458–67. Li YN, Li YY, Wang SX, Ma XY. Efficacy of Bispecific Antibody Targeting EpCAM and CD3 for Immunotherapy in Ovarian Cancer Ascites: An Experimental Study. Curr Med Sci. 2023;43(3):539–50. Xu J, Zhao C, Jia Y, Wang S, Ma X, Wang T, Huang S, Pei M, Wang X, Zhou P. 539P Interim results of a phase I study of M701, a recombinant anti-EpCAM and anti-CD3 bispecific antibody in EpCAM-positive cancer patients with malignant ascites. Ann Oncol. 2021;32:S603. Cai J, Zhang F, Song Z, Jin J, Lv D, Pang W, Yi T, Wang G, Yao J, Wang B, et al. 1371P An anti-EpCAM x CD3 bispecific antibody, M701, for the treatment of malignant pleural effusion in NSCLC patients: Intermediate results of a prospective multicenter phase Ib trial. Ann Oncol. 2024;35:S862. Heiss MM, Murawa P, Koralewski P, Kutarska E, Kolesnik OO, Ivanchenko VV, Dudnichenko AS, Aleknaviciene B, Razbadauskas A, Gore M, et al. The trifunctional antibody catumaxomab for the treatment of malignant ascites due to epithelial cancer: Results of a prospective randomized phase II/III trial. Int J Cancer. 2010;127(9):2209–21. Sebastian M, Kiewe P, Schuette W, Brust D, Peschel C, Schneller F, Rühle KH, Nilius G, Ewert R, Lodziewski S, et al. Treatment of malignant pleural effusion with the trifunctional antibody catumaxomab (Removab) (anti-EpCAM x Anti-CD3): results of a phase 1/2 study. J Immunother. 2009;32(2):195–202. Mau-Sørensen M, Dittrich C, Dienstmann R, Lassen U, Büchler W, Martinius H, Tabernero J. A phase I trial of intravenous catumaxomab: a bispecific monoclonal antibody targeting EpCAM and the T cell coreceptor CD3. Cancer Chemother Pharmacol. 2015;75(5):1065–73. Jacquet P, Sugarbaker PH. Effects of postoperative intraperitoneal chemotherapy on peritoneal wound healing and adhesion formation. Cancer Treat Res. 1996;82:327–35. Lotti M, Giulii Capponi M, Campanati L, Poiasina E, Ansaloni L, Poletti E, Frigerio L. The Onset of Intra-Abdominal Adhesions During Closed-Abdomen Hyperthermic Intraperitoneal Chemotherapy. J Laparoendosc Adv Surg Tech A. 2016;26(12):997–1002. Heiss MM, Strohlein MA, Bokemeyer C, Arnold D, Parsons SL, Seimetx D, et al. The role of relative lymphocyte count as a biomarker for the effect of catumaxomab on survival in malignant ascites patients: results from a phase II/III study. Clin Cancer Res. 2014;20(12):3348–57. Additional Declarations No competing interests reported. Supplementary Files Appendix.docx Cite Share Download PDF Status: Published Journal Publication published 22 Nov, 2025 Read the published version in Experimental Hematology & Oncology → Version 1 posted Editorial decision: Revision requested 25 Aug, 2025 Reviews received at journal 15 Aug, 2025 Reviews received at journal 07 Aug, 2025 Reviewers agreed at journal 07 Aug, 2025 Reviews received at journal 06 Aug, 2025 Reviewers agreed at journal 06 Aug, 2025 Reviewers agreed at journal 05 Aug, 2025 Reviewers agreed at journal 03 Aug, 2025 Reviewers invited by journal 30 Jul, 2025 Editor assigned by journal 24 Jul, 2025 Submission checks completed at journal 24 Jul, 2025 First submitted to journal 24 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7204041","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":494828925,"identity":"baef7f53-fe53-4a32-b1c8-c5bd7023a2a0","order_by":0,"name":"Rongrui Liu","email":"","orcid":"","institution":"Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rongrui","middleName":"","lastName":"Liu","suffix":""},{"id":494828927,"identity":"417d4a43-dcaf-4aa5-b61f-483b96f42f60","order_by":1,"name":"Rongbo Lin","email":"","orcid":"","institution":"Fujian Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rongbo","middleName":"","lastName":"Lin","suffix":""},{"id":494828930,"identity":"d143b737-d910-4a37-a863-698391ee782e","order_by":2,"name":"Ning Li","email":"","orcid":"","institution":"The Affiliated Cancer Hospital of Zhengzhou University \u0026 Henan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Li","suffix":""},{"id":494828932,"identity":"ff17f62f-e4ed-4d4e-8f03-fe0b1a0cf974","order_by":3,"name":"Guiling Li","email":"","orcid":"","institution":"Union Hospital, Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Guiling","middleName":"","lastName":"Li","suffix":""},{"id":494828934,"identity":"fa07f5cf-d463-4558-a4c6-4f6b4200d9b7","order_by":4,"name":"Tao Zhang","email":"","orcid":"","institution":"Union Hospital, Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Zhang","suffix":""},{"id":494828935,"identity":"9948ee32-965b-4b51-9876-e227fa181a5c","order_by":5,"name":"Jun Zhao","email":"","orcid":"","institution":"Changzhi People's Hospital, The Affiliated Hospital of Changzhi Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Zhao","suffix":""},{"id":494828936,"identity":"a641eedd-8318-471e-b4bd-7541cf37bccc","order_by":6,"name":"Jiayi Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Xiamen University, Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Li","suffix":""},{"id":494828937,"identity":"8d270940-8579-429e-bcd1-22f49c2165d3","order_by":7,"name":"Meili Sun","email":"","orcid":"","institution":"Jinan Central Hospital affiliated to Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Meili","middleName":"","lastName":"Sun","suffix":""},{"id":494828938,"identity":"afd961e4-6e83-4c6f-882d-ab0312f21616","order_by":8,"name":"Ke Wang","email":"","orcid":"","institution":"Tianjin Medical University Cancer Institute and Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Wang","suffix":""},{"id":494828939,"identity":"7e9d7f6d-e8c0-4ee2-8787-c22cf9c03881","order_by":9,"name":"Hanxiang An","email":"","orcid":"","institution":"The Cancer Center, Shanxi Bethune Hospital, Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hanxiang","middleName":"","lastName":"An","suffix":""},{"id":494828940,"identity":"2f4fdb28-f69f-4eab-b2fa-c50c4d15c358","order_by":10,"name":"Weijie Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Weijie","middleName":"","lastName":"Zhang","suffix":""},{"id":494828941,"identity":"4bf1d9dd-9763-4a28-a409-270ca5036400","order_by":11,"name":"Huiting Xu","email":"","orcid":"","institution":"Hubei Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huiting","middleName":"","lastName":"Xu","suffix":""},{"id":494828942,"identity":"2b4851ef-ad6c-44d6-b889-394ec3679712","order_by":12,"name":"Shan Zeng","email":"","orcid":"","institution":"Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Zeng","suffix":""},{"id":494828943,"identity":"9742d1a1-f1f8-40bf-aa4d-120eb1f361df","order_by":13,"name":"Mingjun Zhang","email":"","orcid":"","institution":"The second hospital of Anhui medical university","correspondingAuthor":false,"prefix":"","firstName":"Mingjun","middleName":"","lastName":"Zhang","suffix":""},{"id":494828944,"identity":"2d66bba4-b8a6-4afc-b72f-8f4e2d476763","order_by":14,"name":"Wei Duan","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital, Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Duan","suffix":""},{"id":494828945,"identity":"e9c89eef-e39c-4b4d-9bfb-61d6f1f1a7af","order_by":15,"name":"Yuxian Bai","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuxian","middleName":"","lastName":"Bai","suffix":""},{"id":494828946,"identity":"c5122973-70c5-4936-998d-ba055816dc51","order_by":16,"name":"Jingdong Zhang","email":"","orcid":"","institution":"Liaoning Cancer Hospital \u0026 Institute","correspondingAuthor":false,"prefix":"","firstName":"Jingdong","middleName":"","lastName":"Zhang","suffix":""},{"id":494828947,"identity":"4241f29f-06f3-4780-bfaf-828188656990","order_by":17,"name":"He Tian","email":"","orcid":"","institution":"Shandong Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Tian","suffix":""},{"id":494828948,"identity":"1a929d85-bfc0-4ac9-b518-d655e80745a8","order_by":18,"name":"Fei Yin","email":"","orcid":"","institution":"The Fourth Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Yin","suffix":""},{"id":494828949,"identity":"ef8b482a-757d-4e2a-b01b-3861c297fe63","order_by":19,"name":"Yu Kang","email":"","orcid":"","institution":"Obstetrics and Gynecology Hospital of Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Kang","suffix":""},{"id":494828950,"identity":"7c4c006c-8473-4e92-91b1-1483a740bddf","order_by":20,"name":"Qi Xu","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Xu","suffix":""},{"id":494828951,"identity":"081cd335-2d7f-4959-b121-77bf7bc7c492","order_by":21,"name":"Nong Xu","email":"","orcid":"","institution":"The First Affiliated Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Nong","middleName":"","lastName":"Xu","suffix":""},{"id":494828952,"identity":"59baa9ef-9187-4f09-8c5b-429e3f5c5f8e","order_by":22,"name":"Yanhong Deng","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Yanhong","middleName":"","lastName":"Deng","suffix":""},{"id":494828955,"identity":"4ccf9e9a-c0f1-40d5-b5ba-b2cf11ef996c","order_by":23,"name":"Qing Chen","email":"","orcid":"","institution":"Sun Yat-sen Memorial Hospital,Sun Yat-sen,University","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Chen","suffix":""},{"id":494828957,"identity":"371ed840-f5d9-4a9f-93ab-44f3c9497970","order_by":24,"name":"Yongqiang Li","email":"","orcid":"","institution":"Affiliated Cancer Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yongqiang","middleName":"","lastName":"Li","suffix":""},{"id":494828959,"identity":"d2997d9b-1e74-4e4a-988d-7c86f34a28b9","order_by":25,"name":"Hongying Yang","email":"","orcid":"","institution":"Yunnan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongying","middleName":"","lastName":"Yang","suffix":""},{"id":494828963,"identity":"fe6ed6a3-5ec2-4d20-8743-a6dd6e1c1ced","order_by":26,"name":"Fang Su","email":"","orcid":"","institution":"The First Affiliated Hospital of Bengbu Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Su","suffix":""},{"id":494828964,"identity":"db51e01b-346e-40ad-bb57-9ff5848df069","order_by":27,"name":"Zhenghong Xiao","email":"","orcid":"","institution":"Nanshi Hospital of Nanyang","correspondingAuthor":false,"prefix":"","firstName":"Zhenghong","middleName":"","lastName":"Xiao","suffix":""},{"id":494828965,"identity":"2dded552-9b4b-4df3-aeaf-224df1055c02","order_by":28,"name":"Xiaojun Xiang","email":"","orcid":"","institution":"First Affiliated Hospital of Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojun","middleName":"","lastName":"Xiang","suffix":""},{"id":494828967,"identity":"7263c10b-531a-4f9f-bf3b-8619dcec0508","order_by":29,"name":"Pengfei Zhou","email":"","orcid":"","institution":"Wuhan YZY Biopharma Co. Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Pengfei","middleName":"","lastName":"Zhou","suffix":""},{"id":494828968,"identity":"bb24f3d2-6971-437f-bf3e-87ce56dad407","order_by":30,"name":"Shaoyi Huang","email":"","orcid":"","institution":"Wuhan YZY Biopharma Co. Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Shaoyi","middleName":"","lastName":"Huang","suffix":""},{"id":494828970,"identity":"87a746a6-2ec3-466e-b29d-0a7975a2802e","order_by":31,"name":"Jing Zhang","email":"","orcid":"","institution":"Wuhan YZY Biopharma Co. Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":494828972,"identity":"21ea634f-d8d0-42cd-ad1e-06d71e1209f5","order_by":32,"name":"Jianming Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBACxgYg8cFAwo6fmfnwA6K1MM6osEmWbGdLMyDaJmaeM2mMG87zKEgQp3zaGTMJ3rbDzMaHeRgMGGpsogk7bHZamoRk22E+s8O8Bx4wHEvLbSCsJfmYhCHQFrPDfAkGjA2HidGS2CaR2HaYcXMzj4EEkVqAthwAeZ+ZeC1pyZYNwECWOAwM5ARi/GI4O8fw9h9QVPYfPvzgQ40NEVpQVCQQUg4C8sQoGgWjYBSMghEOAG6APgENuLzuAAAAAElFTkSuQmCC","orcid":"","institution":"The Fifth Medical Center, Chinese PLA General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jianming","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2025-07-24 09:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7204041/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7204041/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40164-025-00727-3","type":"published","date":"2025-11-22T15:56:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88339966,"identity":"0499108d-9ffc-4f6e-baf0-794526a96e93","added_by":"auto","created_at":"2025-08-05 12:32:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of M701.\u003c/strong\u003e (a) A schematic diagram and molecular weight (Mw) of M701. M701 consists of a monovalent unit and a single-chain unit. The anti-EpCAM monovalent unit consists of an anti-EpCAM heavy chain and an anti-EpCAM light chain conjugated by a disulfide bond. The anti-CD3 single-chain unit had a VHb-linker-VLb-Fc structure. Two disulfide bonds were formed between the monovalent unit and the single-chain unit. VHa/VLa denotes the variable regions that belong to the monovalent unit, whereas VHb/VLb refers to the variable regions associated with the single-chain unit, and CH3a/CH3b refers to the distinct modifications of the CH3 domain that are introduced to increase heterodimer formation. The modifications include knob-into-hole and salt-bridge. The glycosylated and deglycosylated Mw of each unit of M701 was determined by LC-MS (liquid chromatography-mass spectrometry). G0F is the predominant glycoform inr recombinant human IgG1 antibodies produced in CHO cells. Lys is C-terminal lysine residues at the Fc region of antibodies which is incompletely removed by carboxypeptidase B during antibody production. (b) The affinity of M701 for the anti-EpCAM moiety and the parent anti-EpCAM mAb for HCT116 cells; M701 Kd = 32 nM, and the anti-EpCAM mAb Kd = 16 nM. (c) The affinity of M701 for the anti-CD3 moiety and the parent antibody L2K for human T cells; M701 Kd = 33.7 nM, and L2K Kd = 0.6 nM. (d) M701-mediated cell bridging. The cell bridging of NCI-N87 cells (stained with CFSE) with Cytokine-induced killer cells (CIKs) cells (stained with PKH26) without or with M701 are shown.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7204041/v1/0c7cd02799fd1c100d10f052.png"},{"id":88339230,"identity":"0c642dde-4873-48d7-90c1-92760bbbaab1","added_by":"auto","created_at":"2025-08-05 12:24:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro cytotoxicity and in vivo antitumor efficacy of M701.\u003c/strong\u003e (a) Detection of EpCAM expression in different tumor cell lines via fluorescence-activated cell sorting (FACS). NC was a negative control by using a lymphoma cell line Raji. (b) M701-mediated redirected lysis of cytokine-induced killer cells (CIKs) to six cancer cell lines with different EpCAM expression levels. CIKs were effectors (E) and tumor cells were targets (T). All cell lines were incubated for 24 h in the presence of different concentrations of M701 with CIKs at an E:T ratio of 5:1. M701 inhibited the proliferation of tumor cells HCT116, NCI-N87, KATOIII, SK-OV-3 and MDA-MB-231 with EC50 values of 1.1, 1.8, 8.5, 58.7 and 37.8 ng/mL, respectively. (c) HCT116 xenograft model in CIK-reconstituted NOD/SCID mice treated with M701 (i.v., administered on days 0/2/4). Tumor volumes (day 42, mm³, mean ± SD, n=5): vehicle (1249.89±364.88), anti-EpCAM mAb (827.54±475.95), anti-CD3 isotype (704.03±269.78), M701 2.5 mg/kg (112.54±251.64), 0.5 mg/kg (145.15±124.84), 0.1 mg/kg (828.17±267.18). (d) CT26-mEpCAM ascites model in C57BL/6 mice treated with M708 (i.p., administered on days 0/2/4/7/9/11). M708 is a murine surrogate bsAb (anti-murine EpCAM and murine CD3) of M701. Survival rates (day 46): vehicle (0%), anti-mCD3 isotype 0.25mg/kg (0%), anti-mEpCAM mAb 0.25mg/kg(30%), the combination of anti-mEpCAM mAb 0.25mg/kg and anti-mCD3 isotype 0.25mg/kg (40%), M708 2.5 mg/kg (20%), 0.25 mg/kg (50%), 0.025 mg/kg (60%). Statistics: multiple t-test (c); Log-rank test (d). * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001, ns (not significant). (Abbreviations: bsAb, bispecific antibody; CIK, cytokine-induced killer cell; FACS, fluorescence-activated cell sorting; i.p., intraperitoneal; i.v., intravenous; mAb, monoclonal antibody).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7204041/v1/08ab8a94db289d42f75e05e2.png"},{"id":88339231,"identity":"595baf96-1aa1-4d5e-a1ed-3ce25291c10d","added_by":"auto","created_at":"2025-08-05 12:24:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17936,"visible":true,"origin":"","legend":"\u003cp\u003eCONSORT flow diagram (Only results for the intent-to-treat and safety populations are included in this article. 7 patients in control group transferred to the M701 group after they were intolerant of the ascites.)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7204041/v1/eb40191847fd8a22edf633e2.png"},{"id":88341264,"identity":"05032a61-bce0-459e-b5ae-bd1cb3c24e14","added_by":"auto","created_at":"2025-08-05 12:40:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34131,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier estimates of puncture-free survival. (a) Puncture-free survival in the FAS population. (b) Puncture-free survival in the RLC ≥ 13% population.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7204041/v1/8379d456e77778bbdd3fe519.png"},{"id":88339242,"identity":"c91829ca-0a0d-4c67-9c33-28f369e8b6e1","added_by":"auto","created_at":"2025-08-05 12:24:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":193172,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePuncture-free survival forest plot of the FAS population\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7204041/v1/56e8fcff3f39dd2970cb9a0b.png"},{"id":88339970,"identity":"2610de6f-c8ed-4383-b46d-d7b7cfa032e2","added_by":"auto","created_at":"2025-08-05 12:32:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":47815,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan-Meier estimates of Progression-free Survival and Overall survival. \u0026nbsp;(a) Overall survival in the FAS population; (b) Overall survival in the RLC ≥ 13% subpopulation; (c) Progression-free survival in the FAS population.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7204041/v1/3571a953f2f7175b07cbb771.png"},{"id":96649961,"identity":"f8baa2c9-690e-4f4a-8666-cf5370b59879","added_by":"auto","created_at":"2025-11-24 16:02:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1710385,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7204041/v1/3c0db9b8-d9d7-47bd-897f-b1652580d534.pdf"},{"id":88339234,"identity":"669815b2-d92c-44dc-9b21-b1c62ec8e6f1","added_by":"auto","created_at":"2025-08-05 12:24:21","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19192,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-7204041/v1/89e4684b0933b0e2e911a363.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Clinical Trial of M701, an Anti-EpCAM × Anti-CD3 Bispecific Antibody: A Targeted Intraperitoneal Therapy for Malignant Ascites Stemming from Advanced Solid Tumors","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eMalignant Ascites (MA) refers to the accumulation of fluid in the abdominal cavity due to the presence of various malignant tumors and is most commonly associated with gastrointestinal and gynecological cancers [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The presence of MA indicates that the primary tumor has metastasized either locally or systemically. The build-up of moderate-to-large volumes of fluid often compresses nearby organs, impairing their function. It can also lead to symptoms such as abdominal pain, breathing difficulty, electrolyte imbalances, hypoproteinaemia, and secondary infections [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The prognosis is dire, and the disease imposes a heavy burden on patients, significantly worsening their quality of life [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNo globally approved drug is available that specifically targets the treatment of MA, and no established guidelines or consensus on its diagnosis and management are available [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Clinically, the primary method of symptom relief involves paracentesis or catheter drainage, which offers immediate alleviation. These procedures are often supplemented by treatments, such as diuretics, or the IP administration of chemotherapy or antiangiogenic agents, based on clinical experience. However, these approaches lack robust evidence from clinical trials and have limitations regarding safety and efficacy [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEpithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that functions as an adhesion molecule [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The 39\u0026ndash;42 kDa protein consists of a large extracellular domain with two epidermal growth factor-like repeats, a single transmembrane region, and a short cytoplasmic tail of 26 amino acids [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It is extensively expressed in normal epithelial tissues, including gastric, colon, pancreas, ovarian, and lung tissues, and has low expression levels [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It is frequently overexpressed in various epithelial malignancies and is associated with poor prognosis [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Treatment of head and neck cancer cells, as well as breast cancer cells, with EpCAM-specific antisense oligonucleotides or siRNAs has led to significant reductions, or even complete inhibition, of cell proliferation, migration, and invasion [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, researchers have found EpCAM-positive tumor cells in peritoneal effusions from multiple types of epithelial cancers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, EpCAM is a promising target for treating MA [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eStudies have shown that BsAbs can act in either a combinatorial or an obligate manner, with the latter meaning that the same mechanism of action cannot be achieved by simply combining antibodies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Over the past two decades, T-cell engagers (TCEs) that specifically bind to a tumor surface antigen and the CD3ε chain of the T-cell receptor have dominated this class of bispecific antibodies; several hundred TCEs have been described, with more than 100 advancing to clinical development[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A major challenge in the clinical development of TCEs involves the occurrence of cytokine release syndrome, which is largely caused by the activation of on-target T-cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Although this can often be managed via pretreatment using steroids and by step-up dosing, recent efforts have focused on developing CD3ε antibodies with a decrease in CD3ε affinity to uncouple T-cell killing from cytokine secretion [\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCatumaxomab is a bispecific mouse-rat chimeric monoclonal antibody that targets EpCAM and human cluster of differentiation 3 (CD3). It was approved by the EMA in 2009 for treating MA because of its favorable efficacy in reducing the frequency of puncture or drainage [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, due to commercial reasons, it was withdrawn from the European market in 2017 and reapproved by European Commission in 2025 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ema.europa.eu/\u003c/span\u003e\u003cspan address=\"https://www.ema.europa.eu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This EpCAM\u0026times;CD3 bispecific antibody facilitates the binding of immune cells to tumor cells, boosting the targeted immune response and increasing immune cytotoxicity against tumor cells. Moreover, catumaxomab also binds to CD3 on T cells, triggering their activation and proliferation, causing tumor-killing molecules such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), perforin, and granzyme B to be released [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, catumaxomab exhibits antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity against tumor cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe novel human-mouse chimeric monoclonal antibody M701 targets the same antigens as catumaxomab but features a restructured design and robust CMC properties via the CHO cell expression system. With optimized EpCAM and CD3 affinities, M701 is designed for treating MA and malignant pleural effusions caused by solid epithelial tumors. This bispecific antibody has shown significant inhibitory effects on tumor cells derived from MA in preclinical studies, indicating that it is a targeted therapeutic approach [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. A Phase I clinical study was performed on epithelial tumor patients with MA, and a Phase Ib study was performed on lung cancer patients with malignant pleural effusion. Both studies showed a favorable safety profile and promising efficacy in controlling MA and malignant pleural effusion [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Recently, a Phase II clinical trial was performed to further evaluate the safety and efficacy of IP administration of M701 in patients with advanced epithelial solid tumors complicated by MA.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cb\u003eAnti-EpCAM and anti-CD3 BsAb design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAnti-EpCAM and anti- CD3 BsAb (M701) is a Fab-scFv-Fc bispecific antibody (bsAb), the Fab moiety is anti-EpCAM, and the scFv moiety is anti-CD3. The anti-EPCAM monovalent unit and the anti-CD3 single-chain unit of M701 were from the anti-EpCAM variable regions of AMG110 (Amgen) and L2K, respectively. The mutations in the CH3 domains of the human IgG1 Fc fragment included T366W-Y407A (knobs-into-holes pair), L368R-K409D (ionic bond \u0026ldquo;salt bridge\u0026rdquo;), and D399K-K392D (second salt bridge). The expression system used was CHO-S (Gibco), in which M701 was stably expressed. The purification process was similar to that used to purify classical monoclonal antibodies, including depth filtration, affinity chromatography, low pH, ion-exchange chromatography, nanofiltration, and ultrafiltration/diafiltration (UF/DF).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular weight determination by LC-MS\u003c/b\u003e\u003c/p\u003e\u003cp\u003eReverse-phase HPLC was performed using an ultrahigh-performance system (UPLC Vanquish, Thermo) that was coupled to an orbitrap (QE) mass spectrometer (Q Exactive Plus, Thermo).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell binding assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe HCT116 cells were incubated with serially diluted M701 or HCT116 for 1 h at room temperature and then incubated with PE-conjugated anti-human IgG Fc (secondary antibodies) for 30 min in the dark. The binding activity was evaluated by flow cytometry analysis. The same method was used to measure the affinity of M701 and L2K for human T cells. The dissociation constant Kd was calculated using the software GraphPad Prism. To measure the M701-mediated cell bridging, EpCAM-positive NCI-N87 cells were labeled with 2.5 \u0026micro;M CFSE (Invitrogen), and CD3-positive cytokine-induced killer cells (CIKs) were labeled with 0.2 \u0026micro;M PKH26 (Sigma). The cells were washed three times and mixed at a ratio of 1:1. The mixtures were incubated with serially diluted antibodies (0 to 10 \u0026micro;g/mL) for 30 min at 37\u0026deg;C in a 96-well round bottom plate (Corning). M701-mediated cell bridging was evaluated by conducting flow cytometry analysis and is presented as the percentage of cells in the upper right quadrant of an FL1 vs. FL2 scatter plot, which represents the CFSE-PKH26-double-positive population.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro cytotoxicity assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCIKs were utilized as effector cells to evaluate the in vitro efficacy of M701. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using Ficoll-Hypaque (Sigma) density gradient centrifugation, following the manufacturer\u0026rsquo;s protocol. The isolated PBMCs were then co-cultured with anti-CD3 monoclonal antibody, IFN-γ, IL-2, and IL-1α for 14 days to generate CIKs. The bioactivity of M701 was assessed using a fluorescence-activated cell sorting (FACS)-based cytotoxicity assay. Target cells included EpCAM-high cancer cell lines (HCT116, colorectal carcinoma; OVCAR-3, ovarian adenocarcinoma; KATO-III, gastric carcinoma), EpCAM-low cancer cell lines (SK-OV-3, ovarian adenocarcinoma; MDA-MB-231, breast adenocarcinoma), and the EpCAM-negative cancer cell line U87 (glioblastoma). In the assay, 2.0 \u0026times; 10⁴ CFSE-labeled target cells were co-cultured with CIK effector cells at an effector-to-target (E:T) ratio of 5:1 in 96-well flat-bottom plates (Corning) with serial dilutions of M701 or control antibodies for 24 hours at 37\u0026deg;C in a 5% CO₂ incubator. After incubation, cells were harvested and stained with propidium iodide (PI, Sigma) to assess apoptotic/necrotic populations by flow cytometry.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal studies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMouse Strains and Housing: Female NOD/SCID and C57BL/6 mice (7\u0026ndash;8 weeks old) were purchased from Beijing HFK Bioscience Co., Ltd. Xenograft subcutaneous tumor model: A total of 1.0 \u0026times; 10⁷ HCT116 cells and 1.0 \u0026times; 10⁷ CIK cells were mixed and inoculated subcutaneously into the right dorsal flank of 30 NOD/SCID mice. Within 2 hours of inoculation, the mice were randomly assigned to three experimental groups and three control groups (n\u0026thinsp;=\u0026thinsp;5 per group). Mice in the experimental groups received M701 (0.1, 0.5, or 2.5 mg/kg) via intravenous bolus injection into the lateral tail vein. Mice in the control groups were treated with anti-EpCAM monoclonal antibody (mAb) (2.5 mg/kg), anti-CD3 isotype (2.5 mg/kg), or saline. Treatments were repeated on days 2 and 4. Tumor volumes were measured every three days using a digital caliper, and the tumor volume (mm\u0026sup3;) was calculated using the formula: \u0026frac12; \u0026times; (length \u0026times; width\u0026sup2;).\u003c/p\u003e\u003cp\u003eSyngeneic ascites tumor model: The CT26-mEpCAM cell line, a CT26-derived cell line transfected to express high levels of murine EpCAM, was used in this model. M708, a surrogate of M701, binds both murine EpCAM and murine CD3. The CT26-mEpCAM murine model was established by subcutaneously inoculating 3.0 \u0026times; 10⁶ CT26-mEpCAM cells into the right dorsal flank of C57BL/6 mice. Starting on day 0 and continuing on days 2, 4, 7, 9, and 11, mice received IP bolus injections of 0.025 mg/kg M708, 0.25 mg/kg M708, 2.5 mg/kg M708, 0.25 mg/kg anti-mEpCAM mAb, 0.25 mg/kg anti-mCD3 isotype, a combination of 0.25 mg/kg anti-EpCAM and 0.25 mg/kg anti-mCD3 isotype, or vehicle (n\u0026thinsp;=\u0026thinsp;10 per group). Mice were euthanized when the tumor volume reached 2000 mm\u0026sup3; or at the conclusion of the study.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhase II trial design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe conducted an open-label, multicenter, randomized, and controlled phase II trial, M70102, in 34 hospitals in China between December 2022 and July 2024 (ClinicalTrials.gov identifier: NCT06266091). The study was conducted following the guidelines of the Declaration of Helsinki and approved by the institutional review board of all involved hospitals. All patients provided signed informed consent before joining any study-related procedure.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTrial population\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePatients aged 18\u0026ndash;75 years who were diagnosed with gastrointestinal and ovarian cancers with moderate-to-large-volume symptomatic MA were enrolled. Gastric and colorectal cancer patients had failed at least two lines of systemic treatment, and ovarian cancer patients were platinum-resistant. Additionally, only patients with an Eastern Cooperative Oncology Group (ECOG) performance status of 0\u0026ndash;2, life expectancy\u0026thinsp;\u0026ge;\u0026thinsp;8 weeks and adequate bone marrow function, defined as absolute neutrophil count\u0026thinsp;\u0026ge;\u0026thinsp;1.5 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e/L, platelet count\u0026thinsp;\u0026ge;\u0026thinsp;80 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e/L, hemoglobin\u0026thinsp;\u0026ge;\u0026thinsp;9.5 g/dL, serum albumin\u0026thinsp;\u0026ge;\u0026thinsp;28 g/L, and relative lymphocyte count (RLC)\u0026thinsp;\u0026ge;\u0026thinsp;10% in peripheral blood, were allowed to participate. Patients whose serum creatinine level was \u0026gt;\u0026thinsp;1.5 times the upper limit of normal were excluded. A washout period of two weeks before cycle 1, day 1 was mandatory if the patients were administered any IP chemotherapy.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTrial intervention\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe patients were randomly assigned to two groups at a ratio of 1:1. In the M701 group; patients received paracentesis and IP infusions of 50, 400, 400, and 400 \u0026micro;g of M701 on days 1, 4, 11, and 18, respectively. Additional M701 IP infusions could be administered every two weeks without requiring punctures or drainage until the MA was intolerant or patients died. In the control group, patients received paracentesis alone as needed from days 1 to 18. Both arms received systemic tumor treatment as determined by investigators following the guidelines. After day 18, no puncture or drainage was allowed for the patients in either arm until the MA was intolerant. The patients in the control group were allowed to transfer to the M701 group and receive the M701 IP infusion after they reached the major endpoint.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEndpoints and statistical analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe primary endpoint was PuFS, defined as the time to the first repuncture or drainage due to intolerance of MA or death after the last drainage from days 1 to 18, whichever occurred first. The criteria for ascites intolerance include: 1) The patient complains that he/she cannot tolerate the ascites; 2) The presence of a large amount of ascites in the patient is determined by B-ultrasound (the maximum depth of ascites\u0026thinsp;\u0026ge;\u0026thinsp;4.5 cm); 3) The investigator determines that the patient's total score for ascites symptoms and signs exceeds 7 points (by Likert 4-points scale, see Appendix 1). The secondary efficacy endpoints included OS, progression-free survival (PFS) for the target lesion according to the RECIST 1.1 criteria, and the best objective response rate of MA following the WHO criteria.\u003c/p\u003e\u003cp\u003eSafety was assessed based on adverse events reported during the study. All adverse events were coded according to Medical Dictionary for Regulatory Activities (MedDRA 27.0) and graded according to the Common Terminology Criteria for Adverse Events (CTCAE) V5.0. Cytokine release syndrome (CRS) is the only listed adverse event of special interest (AESI).\u003c/p\u003e\u003cp\u003eThe sample size was determined by conducting log-rank tests. The assumption was that the median PuFS of the control group was 18 days, and the hazard ratio (HR) between the experimental group and the control group was 0.5. With a power of 80%, alpha\u0026thinsp;=\u0026thinsp;5%, and a dropout rate of 10%, 60\u0026ndash;80 patients were needed.\u003c/p\u003e\u003cp\u003eFisher\u0026rsquo;s exact test was conducted to analyze nominal variables, and the Mann-Whitney U test was conducted to analyze continuous variables. The survival curves were estimated using the Kaplan-Meier method. All data were analyzed using Statistical Analysis System software (SAS\u0026reg; 9.4 or higher version).\u003c/p\u003e\u003cp\u003eTo conduct cell and animal studies, statistical analysis was performed using Prism 6 (GraphPad Software Inc., La Jolla, CA). The differences between groups were determined by conducting two-sided Student\u0026rsquo;s t-tests. All data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. All results were considered to be statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePharmacokinetics and immunogenicity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBlood and ascites samples were collected to evaluate the pharmacokinetics and immunogenicity of M701. The pharmacokinetic analysis set (PKAS) and immunogenicity analysis set (IS) included all patients who received at least one dose of M701 infusion and from whom adequate drug concentration or anti-drug antibody (ADA) measurements were obtainable during the trial. Blood samples for pharmacokinetic and immunogenicity analysis were collected on days 1, 4, 18, 46, and 88 and every two treatment cycles 1 h before M701 infusion. Ascites samples for PK and immunogenicity analysis were collected on days 1, 18, 32, 46, and 88 and every two treatment cycles 24 h before M701 infusion. The serum and ascites concentrations of M701 or ADA were determined using validated methods.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eCharacteristics of M701\u003c/b\u003e\u003c/p\u003e\u003cp\u003eM701 consists of two distinct units, including a monovalent unit comprising a heavy chain/light chain pair and a single-chain unit (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In the single-chain unit, the single-chain variable fragment (scFv) is constructed in a variable heavy chain (VH)-variable light chain (VL) orientation, with VH and VL connected in-frame by a (Gly\u003csub\u003e4\u003c/sub\u003eSer)\u003csub\u003e3\u003c/sub\u003e linker, and the scFv is fused to the human IgG1 Fc fragment to form the single-chain This design prevents mismatched pairing and increases the manufacturability of the antibody.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe glycosylated and deglycosylated intact mass of M701 was 128.5kDa and 125.6kDa. Under reducing conditions, the molecular weight (Mw) of the light chain was 24.1 kDa, the glycosylated Mw of the heavy chain was about 50.8 kDa and the single-chain was 53.6 kDa, the deglycosylated Mw of the heavy chain was about 49.4 kDa, and the single-chain was 52.1 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eThe mutations in the Fc fragments of M701 were based on a salt bridge and knobs-into-holes (KIHs). The specific modifications were T366W, K392D, and K409D on the Fc of monovalent unit and L368R, D399K, and Y407A on the Fc of single-chain unit.\u003c/p\u003e\u003cp\u003e\u003cb\u003eM701 exhibited moderate affinity and dose-dependent cell bridging\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cell binding activity of M701 was evaluated by conducting flow cytometry assays. The results indicated that the affinity of M701 (Kd\u0026thinsp;=\u0026thinsp;32 nM) was about two times weaker than that of the anti-EpCAM mAb (Kd\u0026thinsp;=\u0026thinsp;16 nM) for HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and significantly weaker than that of L2K for human T cells (M701 Kd\u0026thinsp;=\u0026thinsp;33.7 nM, L2K Kd\u0026thinsp;=\u0026thinsp;0.6 nM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Additionally, the mechanism of M701-mediated recruitment of CD3-positive cells to EpCAM-positive cells was investigated. CIKs expressing CD3 were labeled with PHK26, and EpCAM-positive NCI-N87 cells were labeled with CFSE. The proportion of double-positive cells was less than 10% in the absence of M701 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In contrast, in the presence of 10 \u0026micro;g/mL M701, up to 40% of the total cell population was double-positive for M701-mediated cell bridging. The enhanced level of cell bridging mediated by M701 was also dose-dependent. Neither the anti-EpCAM mAb nor L2K could mediate the cell bridging at any concentration tested.\u003c/p\u003e\u003cp\u003e\u003cb\u003eM701 potently redirected lysis to EpCAM-positive tumor cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe evaluated the cytotoxic effects of M701, mediated by CIKs, on cancer cells with varying levels of EpCAM expression. Target cells with different EpCAM expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) were incubated with increasing concentrations of M701 and CIKs at an E:T ratio of 5:1. M701 showed potent cytotoxicity against HCT116, NCI-N87, and KATO III cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), all of which express high levels of EpCAM. However, M701 displayed minimal cytotoxic effects on SK-OV-3 and MDA-MB-231 cells, which express very low levels of EpCAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Additionally, M701 did not induce cytotoxicity in EpCAM-negative U87 cells, confirming the specificity of the M701-mediated cell lysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eM701 specifically induces potent cytotoxicity in EpCAM-positive tumor cells, with minimal effects on EpCAM-negative cells, highlighting its potential as a targeted therapeutic agent for EpCAM-expressing cancers.\u003c/p\u003e\u003cp\u003e\u003cb\u003eM701 and surrogate M708 exhibited potent antitumor efficacy in solid and ascites tumor models\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn a xenograft subcutaneous tumor model, M701 demonstrated superior efficacy in inhibiting the growth of human colon cancer cells (HCT116) in NOD-SCID mice compared to control antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eAs a surrogate of M701, M708 possesses the same Fab-scFv-Fc structure and binds both murine EpCAM and murine CD3. In a syngeneic ascites tumor model, IP injection of M708 significantly prolonged the survival of mice with malignant ascites at low, medium, and high doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eThese indicates that a bsAb targeting EpCAM and CD3 has a strong inhibitory effect on EpCAM-positive tumors and the malignant ascites caused by these tumors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBaseline characteristics of patients in the Phase II trial\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrom November 2021 to July 2024, 84 patients were enrolled, 43 patients were assigned to the M701 group, 41 patients were assigned to the control group, and one patient in the control group withdrew from the study after initial screening. In total, 83 patients who were administered at least one M701 infusion in the M701 group or one therapeutic paracentesis in the control group were included in the full analysis set (FAS). According to the protocol, seven patients in the control group were transferred to the M701 group to receive M701 via IP infusion. The CONSORT flow diagram are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The baseline characteristics of the intent-to-treat (ITT) population are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and the patients and disease characteristics were balanced between the M701 group and the control group. Most patients in both arms had gastric cancer, accounting for 48.8% of those in the M701 group and 45.0% of those in the control group. The percentage of patients with prior paracentesis was 65.1% in the M701 group and 65.0% in the control group, and the median number of patients with prior paracentesis was one and two in the M701 and control groups, respectively. The median number of prior lines of systemic treatment in both groups was two. A total of 30.2% and 32.5% of patients in the M701 and control groups, respectively, received more than three lines of systemic treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePatient characteristics in ITT (N\u0026thinsp;=\u0026thinsp;84)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eCharacteristic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eITT Set\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM701\u003c/p\u003e\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;43)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;41)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eAge(years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean (SD)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54.8 (8.99)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.4 (10.64)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedian (P25,P75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54 (50.0\u0026ndash;59.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54 (49.0-62.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 (23%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14 (34%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14 (33%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14 (34%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29 (67%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27 (66%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eECOG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (5%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34 (80%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e34 (83%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (9%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 (12%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eCancer Type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGastric\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21 (49%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19 (46%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOvarian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13 (30%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13 (32%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColorectal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 (19%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8 (20%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFallopian tube cancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (2%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimary peritoneal cancer,\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eIf maintain the system treatment regimen after enrolled\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 (7%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3 (7%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40 (93%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38 (93%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eClinical Stages\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (2%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (2%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 (23%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3 (7%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30 (70%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37 (90%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (5%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePrevious treatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIP treatments\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24 (56%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21 (51%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eParacentesis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27 (63%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25 (61%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003ePrevious paracentesis frequency (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e37.2%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e46.3%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u0026ndash;3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.2%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31.7%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;4 times\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.1%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnknown\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.9%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment outcomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the efficacy of M701 in controlling MA, PuFS was set as the primary endpoint and directly reflected the ability of treatment to prevent the reaccumulation of ascites in the peritoneal cavity. Median PuFS was significantly longer in the M701 group in the ITT population (75 vs. 25 days) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), with an HR of 0.43 (95% CI: 0.22, 0.81) and p\u0026thinsp;=\u0026thinsp;0.0065. The one-month PuFS rates were 65.2% in the M701 group and 40.6% in the control group, whereas the two-month PuFS rates were 50.3% in the M701 group and only 22.1% in the control group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A significant difference in the two-month PuFS rates was found between the two groups (p\u0026thinsp;=\u0026thinsp;0.001), indicating that compared to the control group, the M701 group had a significantly longer time without the need for peritoneal drainage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePuFS in the FAS and RLC\u0026thinsp;\u0026ge;\u0026thinsp;13% of populations\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eFAS population\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eRLC\u0026thinsp;\u0026ge;\u0026thinsp;13% population\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM701\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eM701\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedian PuFS (95%CI), Day\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e75 (29\u0026ndash;153)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25 (12\u0026ndash;32)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e125 (45\u0026ndash;174)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22 (11\u0026ndash;32)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value (Log-rank test)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.0065\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.0003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHazard Ratio(95%CI)\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.43 (0.22, 0.81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.23(0.10, 0.55)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHalf a month PuFS rate (95%CI)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e80.8\u003c/p\u003e\u003cp\u003e(65.2, 89.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e67.2\u003c/p\u003e\u003cp\u003e(49.5, 79.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e86.6\u003c/p\u003e\u003cp\u003e(68.1, 94.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e63.1\u003c/p\u003e\u003cp\u003e(44.3, 77.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1-month PuFS rate (95%CI)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e65.2\u003c/p\u003e\u003cp\u003e(47.1, 78.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40.6\u003c/p\u003e\u003cp\u003e(23.6, 56.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e73.4\u003c/p\u003e\u003cp\u003e(51.5, 86.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e38.2\u003c/p\u003e\u003cp\u003e(21.1, 55.2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-month PuFS rate (95%CI)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50.3\u003c/p\u003e\u003cp\u003e(31.8, 66.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.1\u003c/p\u003e\u003cp\u003e(8.2, 40.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e67.8\u003c/p\u003e\u003cp\u003e(44.7, 82.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18.3\u003c/p\u003e\u003cp\u003e(5.5, 37.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003ea. Based on the Kaplan-Meier estimates; b. Based on the Log-rank test; c.Based on the Cox regression.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe PuFS in different subpopulations was also analyzed. The different subpopulations benefited from M701 treatment, regardless of sex, age, type of cancer, clinical stage, previous treatment history, and time point of disease diagnosis. Compared to the women in the control group, women with Stage IV disease who previously received local chemotherapy or paracentesis significantly benefited from M701 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAn in vitro study revealed that M701 could bridge T lymphocytes to EpCAM-positive tumor cells, which activated T lymphocytes to kill tumor cells. Therefore, the rationale that patients with high T lymphocyte counts respond better to M701 treatment was verified in the subpopulation with a high RLC in whole blood cells. In the population with RLC\u0026thinsp;\u0026ge;\u0026thinsp;13% at baseline, the M701-treated patients showed an even greater PuFS benefit than individuals in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The median PuFS times were 125 days and 22 days, respectively (p\u0026thinsp;=\u0026thinsp;0.0003, HR\u0026thinsp;=\u0026thinsp;0.23 (0.10, 0.55)) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTumor response to systemic therapy was not found in either group (Objective response rate\u0026thinsp;=\u0026thinsp;0%). The PFS in those two groups was not significantly different (HR\u0026thinsp;=\u0026thinsp;0.80 and p\u0026thinsp;=\u0026thinsp;0.40) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe final analysis of survival suggested that treatment with M701 provided a trend of survival benefit with HR\u0026thinsp;=\u0026thinsp;0.68 and p\u0026thinsp;=\u0026thinsp;0.1443 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The six-month survival rate in the M701 group was considerably higher than that in the control group (33.3% vs 12.1%) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the population with RLC\u0026thinsp;\u0026ge;\u0026thinsp;13% at baseline, the M701-treated patients received significant benefits in overall survival (HR\u0026thinsp;=\u0026thinsp;0.46 and p\u0026thinsp;=\u0026thinsp;0.0155) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb)\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOverall survival in the FAS and RLC\u0026thinsp;\u0026ge;\u0026thinsp;13% of populations\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eFAS population\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eRLC\u0026thinsp;\u0026ge;\u0026thinsp;13% population\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM701\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eM701\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedian OS(95%CI), Daya\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e110 (82, 177)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76 (55, 116)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e152 (94, 244)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e76 (55, 116)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP value(Log-rank test)b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.1443\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.0155\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrata Hazard Ratio (95%CI)c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0.68 (0.40, 1.15)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.46 (0.24, 0.88)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-month survival rate(95%CI)a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e75.9 (59.9, 86.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66.7 (47.9, 80.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e86.5 (67.9, 94.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e69.0 (48.8, 82.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-month survival rate(95%CI)a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58.8 (42.3, 72.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45.5 (28.2, 61.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72.7 (52.7, 85.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e44.8 (26.5, 61.6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6-month survival rate(95%CI)a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e33.3 (19.4, 47.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.1 (3.8, 25.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40.1 (22.3, 57.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.8 (4.3, 28.6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003ea. Based on the Kaplan-Meier estimates; b. Based on the Log-rank test; c. Based on the Cox regression; d. The transferred patients in the control group were excluded.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eM701 exposure and Safety profiles\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAmong the patients in the M701 group and the control group transferred to the M701 group, 50 patients received between 1 and 16 infusions of M701 individually. The median number of infusions was four, with an average of five infusions per patient.\u003c/p\u003e\u003cp\u003eAll patients in the control group received a puncture and an indwelling catheter. Among them, 85% carried two or more drainages from days 1 to 18.\u003c/p\u003e\u003cp\u003eAlthough the IP infusion of M701 is a kind of local therapy, it is hypothesized that it will increase the risk to patients because of T lymphocyte activation. In this study, 50 subjects received at least one dose of M701 and systemic therapy, while 40 subjects in the control group received systemic therapy and paracentesis. Treatment emergent adverse events (TEAEs) occurred in 47 subjects (94.0%) in the experimental group and 37 subjects (92.5%) in the control group, with similar TEAE incidence rates between the two groups. The incidence of \u0026ge;\u0026thinsp;Grade 3 TEAE occurred in 52.0% and 55.0% of the subjects in the two groups, respectively. The results showed that 50% of subjects in both groups encountered SAEs. The details of the safety profile are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Compared to paracentesis alone, the local infusion of M701 combined with systemic therapy did not cause risk to patients.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOverall adverse events in SS\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM701\u003c/p\u003e\u003cp\u003e(N\u0026nbsp;=\u0026nbsp;50)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003cp\u003e(N\u0026nbsp;=\u0026nbsp;40)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCase (incidence %)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCase (incidence %)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTEAE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e47(94.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e37(92.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM701 related AE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40 (80.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSystemic therapy related AE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36 (72.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30 (75.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026ge; Grade 3 TEAE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e26 (52.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22(55.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u0026ge; Grade 3 M701 related AE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e24 (48.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSAE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25(50.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20(50.0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCRS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2 (4.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn the M701 group, the most common SAEs included gastrointestinal disorders (16.0%), laboratory examinations (14.0%), blood and lymphatic system disorders (12.0%), infections and infestations (10.0%), general disorders and administration site conditions (6.0%), and disorders related to metabolism and nutrition (6.0%). In the control group, the most common SAEs included gastrointestinal disorders (22.5%), infections and infestations (15.0%), disorders related to metabolism and nutrition (10.0%), and general disorders and administration site conditions (4.0%). The details of the SAEs are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of patient SAEs in the safety set (\u0026ge;\u0026thinsp;5%)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eM701 group\u003c/p\u003e\u003cp\u003e(N\u0026nbsp;=\u0026nbsp;50)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControl group\u003c/p\u003e\u003cp\u003e(N\u0026nbsp;=\u0026nbsp;40)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003cp\u003e(N\u0026nbsp;=\u0026nbsp;90)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBy SOC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCases/Incidence(%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCases/Incidence(%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCases/Incidence(%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePatients with \u0026ge;\u0026thinsp;1 SAE event\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e25 (50.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e20 (50.0 )\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e45 (50.0 )\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGastrointestinal disorders\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e8 (16.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e9 (22.5)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e17 (18.8)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIntestinal obstruction\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3(6.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1(2.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4(4.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLab examinations\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e7(14.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e1(2.5)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e8(8.9)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWhite blood cell count decreased\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3(6.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3(3.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlatelet count decreased\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3(6.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3(3.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNeutrophil count decreased\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3(6.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3(3.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBlood and lymphatic system disorders\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e6 (12.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e6 (6.7)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnemia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 (10.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 (5.5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eInfections and infestations\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e5 (10.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e6 (15.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e11 (12.2)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGeneral disorders and administration site conditions\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e3(6.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e2 (5.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e5 (5.6)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMetabolism and nutrition disorders\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e3(6.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e4(10.0)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e7 (7.8)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDecreased appetite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2(4.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3(7.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5(5.6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCompared to the individuals in the control group, the patients in the M701 group had a greater incidence of SAE in terms of decreased blood cells and anemia, which may be related to the activation of T-lymphocytes. However, those SAEs in blood cells were manageable and reversible. No patients discontinued the M701 treatment because of severe adverse reactions. The ASEI in this study was CRS which were frequently reported in TCEs treated patients. CRS occurred in 4% of the M701 group and the CTCAE grades ranged from 1 to 2.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePharmacokinetics and immunogenicity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAmong the 50 patients who received at least one infusion of M701, 42 had at least one blood or ascites sample collected for pharmacokinetics and immunogenicity analysis. These patients were included in the PK analysis set and immunogenicity set. The M701 concentration was below the lower limit of quantitation in most of the serum and ascites samples. The highest serum concentration of M701 was found on day 18, and the arithmetic mean was 6.53 ng/mL. The highest concentration of M701 in ascites was found on day 18, and the arithmetic mean was 26.5 ng/mL.\u003c/p\u003e\u003cp\u003eIn the serum, the rate of ADA positivity was 64.3%, which was caused by the M701 treatment, whereas in the ascites, it was 45.2%. As the number of administrations increased, the titer of ADA increased, both in the serum and ascites. However, the ADA titer in ascites was considerably lower than that in serum, and the ratio was about 0.1\u0026ndash;0.3.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eMA has been a great challenge for cancer patients and oncologists for decades. Although new drugs for systemic cancer treatment have emerged in the last two decades, significantly prolonging the survival of cancer patients, the issue of MA has become more prominent for patients with advanced-stage cancer. Different drugs or devices have been tested in many clinical trials, including TNFα, IL-2, chemotherapy, filtered ascites, and catumaxomab, among which only catumaxomab completed a controlled randomized clinical trial and was approved by the EMA for MA treatment in 2009[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Due to certain commercial reasons, Catumaxomab was withdrawn from the European market in 2017. However, after eight years, no new, more effective treatments have emerged on the market. As a result, in 2025, the European Commission re-approved the marketing of catumaxomab in the European Union for the treatment of malignant ascites, based on previous clinical research results.\u003c/p\u003e\u003cp\u003eIn this study, M701 was constructed on an asymmetric bispecific antibody platform (YBODY\u0026reg;) developed by YZYBIO, Inc., with favorable characteristics concerning pairing efficacy, yield, and stability using the CHO cell expression system. In vitro assays revealed that M701 specifically trafficked CD3-positive cells to EpCAM-positive cells and caused lysis of only tumor cells with moderate-to-high EpCAM expression, indicating that M701 has few side effects on normal tissue. In xenograft tumor-bearing mice, M701 inhibited tumor growth but neither the anti-EpCAM antibody nor the anti-CD3 antibody performed such inhibition. In an immunocompetent C57BL/6 mouse model, a surrogate molecule targeting murine EpCAM and CD3 also prolonged the survival of mice with MA.\u003c/p\u003e\u003cp\u003eTo evaluate the safety and efficacy of M701, this phase II trial was conducted on advanced cancer patients with moderate-to-large-volume symptomatic MA who had failed at least two lines of systemic treatment or were platinum-resistant. The patients in the M701 group received an IP infusion of M701 after drainage, whereas the patients in the control group received only drainage. Moreover, both groups received the systemic treatments selected by the investigator based on the Chinese clinical guidelines. The results of the safety evaluation revealed that these combinations were well-tolerated and that local infusion of M701 did not increase the risk to patients. The highest incidence of M701-related SAEs was the abnormal level of all kinds of blood cells, including decreased white blood cells, platelets, and anemia, which may be due to the activation of T lymphocytes. However, those AEs were transient and reversible. No patient in the M701 group discontinued treatment because of abnormal laboratory examinations. Owing to its asymmetric structure and human-mouse chimerism, M701 has a moderate CD3 affinity and low immunogenicity, which indicates that M701 has the innate characteristics of low incidence of CRS and human anti-mouse antibody (HAMA) reaction. Therefore, the IP infusion of M701 was performed more than 10 times without causing strong CRS or HAMA. Additionally, hepatic toxicity, which is a common ADR caused by catumaxomab, was not observed in M701-treated patients [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Moreover, repeated M701 IP infusions did not cause prominent abdominal compartment separation, which is frequently reported in patients who have undergone IP chemotherapy [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRegarding effectiveness in controlling MA, the M701 group showed an encouraging effect, with a median PuFS of 75 days, which was better than that of the control group (25 days) and the historical data of a pivotal trial of catumaxomab (46 days) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The extra benefit may come from systemic treatment since the control group in this study had a longer PuFS than the historical data of the control group in the catumaxomab pivotal study (11 days) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]or the lower grade and incidence of CRS and relative adverse drug reactions. The patients in the control group in this study had more drainage than those in the catumaxomab pivotal study (only once); this fact may also have contributed to the prolonged PuFS in the control group. Systemic chemotherapy or antiangiogenic drugs may also weaken hematopoietic function, which may decrease the effect of T-cell-engaging antibodies and increase the risk of M701 infusion. Therefore, we could only conclude that M701 infusion significantly prolonged the puncture/drainage interval with regular systemic treatments.\u003c/p\u003e\u003cp\u003eAccording to the analysis of PuFS in the subpopulation, some subpopulations, including female patients and patients who previously underwent chemotherapy or paracentesis, received significant benefits.\u003c/p\u003e\u003cp\u003eAs a local treatment, M701 infusion is unlikely to improve OS because of the low dose of M701 (400 \u0026micro;g) and limited distribution in peripheral blood. No objective response was found during this phase II study in either the M701 or the control group (data not shown). However, the M701 group showed trends of longer OS in the FAS population and most subpopulations. Gastrointestinal cancer patients have longer OS than ovarian cancer patients, which is consistent with the results of pivotal trials of catumaxomab in MA patients. The increase in OS may be due to improvements in the physical status or willingness to continue systemic treatment of patients with a heavy burden of MA.\u003c/p\u003e\u003cp\u003eSince M701 kills tumor cells in the peritoneal cavity by recruiting and activating T lymphocytes, we hypothesized that patients with higher lymphocyte counts at baseline may receive greater benefits from M701 treatment. The results of the study revealed that patients with \u0026ge;\u0026thinsp;13% RLC in peripheral blood had better PuFS than the FAS population (125 days vs. 75 days). These patients also showed significantly longer OS (152 days vs 76 days). Over 40% of patients in this subgroup survived for six months, whereas most MA patients were found to survive for only 2\u0026ndash;3 months [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This trend was also observed in the pivotal trial of catumaxomab as well [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e In the phase II study of M701 in MA patients, the participants in both groups received the systemic treatment recommended by the Chinese clinical guidelines for gastrointestinal cancer patients who had failed two lines of chemotherapy and for ovarian cancer patients who were resistant to platinum-based chemotherapy. Systemic treatments may affect PuFS and OS, however, considering that the allocation of tumor types and the number of previous treatment lines in the two groups were balanced, the bias caused by systemic therapies should be minimal.\u003c/p\u003e\u003cp\u003eMoreover, there are still certain limitations in this study, including: 1) the sample size is relatively small, and the degree of benefit for patients with different types of tumors has not yet been determined; 2) the QoL data at different time points were not effectively collected. Therefore, further validation of the clinical value of M701 is needed in larger and more well-designed clinical studies.\u003c/p\u003e\u003cp\u003eGiven its promising efficacy and safety profile, multiple-center pivotal stage III clinical research is ongoing to test the efficacy and safety of M701 in China. Further clinical studies are planned with patients outside China. The importance and benefits of repeated infusion of M701 or rechallenge with M701 after MA intolerance require further evaluation in those clinical trials.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMalignant Ascites\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePuFS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePuncture-free survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOverall Survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSAE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSerious adverse event\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTEAE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTreatment emergent adverse event\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAESI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAdverse event of special interest\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEpCAM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEpithelial cell adhesion molecule\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTCEs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eT-cell engagers\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCD3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCluster of differentiation 3\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCRS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCytokine release syndrome\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTNF-\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTumor necrosis factor-alpha\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIFN-\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterferon-gamma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ebsAb\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ebispecific antibody\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCIKs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCytokine-induced killer cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eECOG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEastern Cooperative Oncology Group\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRLC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRelative lymphocyte count\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIntraperitoneal\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePFS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eProgression-free Survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCTCAE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCommon Terminology Criteria for Adverse Events\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHazard ratio\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eADA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAnti-drug antibody\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMw\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMolecular weight\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKIH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKnobs-into-hole\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFAS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFull analysis set\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHAMA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHuman anti-mouse antibody\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to thank all subjects and their families who participated in this study and all investigators and research personnel in those study centers.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eConception and design: J.X. and P.Z.; development of methodology: R.L., J.Z., S.H. and J.X; investigation, resources, and data curation: R.L., R.L., N.L., G.L., T.Z., J.Z, J.L., M.S., K.W., H.A., W.Z., H.X., S.Z., M.Z., W.D., Y.B., J.Z., H.T, F.Y., Y.K., Q.X., N.X., Y.D., Q.C., Y.L., H.Y., F.S., Z.X., X.X, and J.X.; analysis and interpretation of data: R.L., J.Z., S.H. and J.X.; writing (review and editing): R.L., J.Z., S.H. and J.X.; study supervision: J.X. and P.Z.. All authors contributed to manuscript editing and approved the submission of the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was completely sponsored by Wuhan YZY Biopharma Co. Ltd.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe clinical study was conducted according to the Declaration of Helsinki and was approved by the institutional review board of each hospital. Informed consent was taken from all patients before participating in any study-related procedure. Animal Studies were approved by Huazhong University of Science and Technology Experimental Animal Ethics Committee.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis manuscript has not been previously published and is not under consideration for publication elsewhere.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eDr. Pengfei Zhou, Dr. Shaoyi Huang and Dr. Jing Zhang are the employees of Wuhan YZY Biopharma Co. Ltd, which sponsored this study and owned the commercial development rights of M701.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026rsquo; detail\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Oncology, The First Medical Center, Chinese PLA General Hospital,28 Fuxing Road, Beijing 100039, China; \u003csup\u003e2\u003c/sup\u003eDepartment of Abdominal Oncology, Fujian Cancer Hospital, No.420 Fuma Road, Fuzhou 350000, China; \u003csup\u003e3\u003c/sup\u003eDepartment of Gastrointestinal Oncology, The Affiliated Cancer Hospital of Zhengzhou University \u0026amp; Henan Cancer Hospital, No.127 Dongming Road, Zhengzhou 450000, Henan, China; \u003csup\u003e4\u003c/sup\u003eDepartment of Gynecologic Oncology, Cancer Center of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefang Avenue, Wuhan 430000, Hubei, China; \u003csup\u003e5\u003c/sup\u003eDepartment of Abdominal Oncology, Cancer Center of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefang Avenue, Wuhan 430000, China; \u003csup\u003e6\u003c/sup\u003eDepartment of Oncology, Changzhi People\u0026apos;s Hospital, The Affiliated Hospital of Changzhi Medical College, No.502 Changxing Middle Road, Changzhi \u0026nbsp;046000, China; \u003csup\u003e7\u003c/sup\u003eDepartment of Medical Oncology, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, No.55 Zhenhai Road, Xiamen 361005, China; \u003csup\u003e8\u003c/sup\u003eDepartment of Oncology, Jinan Central Hospital affiliated to Shandong University, No.105 Jiefang Road, Jinan 250013, China; \u003csup\u003e9\u003c/sup\u003eDepartment of Gynecological Oncology, Tianjin Medical University Cancer Institute and Hospital, West Huan-Hu Road, Tianjin300060, China; \u003csup\u003e10\u003c/sup\u003eDepartment of Gastrointestinal Oncology, The Cancer Center, Shanxi Bethune Hospital, Shanxi Medical University, No.99 Longcheng Street,Taiyuan 030032, China; \u003csup\u003e11\u003c/sup\u003eDepartment of Oncology, The First Affiliated Hospital of Zhengzhou University, No.1 Jianshe Dong Road, Zhengzhou 450052, China; \u003csup\u003e12\u003c/sup\u003eDepartment of Medical Oncology, Hubei Cancer Hospital, No 116 Zhuodaoquan South Road, Wuhan 430079, China; \u003csup\u003e13\u003c/sup\u003eDepartment of Chemotherapy Oncology, Xiangya Hospital of Central South University, No.87 Xiangya Road, Changsha 410008 , China; \u003csup\u003e14\u003c/sup\u003eDepartment of Oncology, The second hospital of Anhui medical university, No.678 Furong Road, Hefei 230601, China; \u003csup\u003e15\u003c/sup\u003eDepartment of Gynecologic Oncology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, No.17 Qihelou, Beijing 100069, China; \u003csup\u003e16\u003c/sup\u003eDepartment of Gastrointestinal Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin150040, China; \u003csup\u003e17\u003c/sup\u003eDepartment of Gastroenterology, Liaoning Cancer Hospital \u0026amp; Institute,No.44 Xiaoheyan Road, Shenyang 110042, China; \u003csup\u003e18\u003c/sup\u003eDepartment of Gastroenterology, Shandong Cancer Hospital, No.440 Jiyan Road, Jinan 250117, China; \u003csup\u003e19\u003c/sup\u003eDepartment of Gastroenterology, The Fourth Hospital of Hebei Medical University, No.12 Jiankan Road, Shijiazhuang 050011, China; \u003csup\u003e20\u003c/sup\u003eDepartment of Gynecological Oncology, Obstetrics and Gynecology Hospital of Fudan University,No.419 Fangxie Road, Shanghai 200011, China; \u003csup\u003e21\u003c/sup\u003eDepartment of Hepato-Pancreato-Biliary \u0026amp; Gastric Medical Oncology, Zhejiang Cancer Hospital, No. 1 East Banshan Road, Hangzhou 310022, China; \u003csup\u003e22\u003c/sup\u003eDepartment of Oncology, The First Affiliated Hospital, Zhejiang University School of Medicine,No.79 Qingchun Road, Hangzhou 310003, China; \u003csup\u003e23\u003c/sup\u003eDepartment of Oncology, The Sixth Affiliated Hospital, Sun Yat-sen University, No. 26 Yuancun Er Heng Road, Guangzhou 510655, China; \u003csup\u003e24\u003c/sup\u003eDepartment of Obstetrics and Gynecology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, No.107 Yanjiang Road West, Guangzhou 510120, China; \u003csup\u003e25\u003c/sup\u003eThe First Department of Chemotherapy, Affiliated Cancer Hospital of Guangxi Medical University, 71 Hedi Road, Nanning 530021, China; \u003csup\u003e26\u003c/sup\u003eDepartment of Gynaecology, Yunnan Cancer Hospital, No.519 Kunzhou Road, Kunming 650118, China; \u003csup\u003e27\u003c/sup\u003eDepartment of Oncology, The First Affiliated Hospital of Bengbu Medical University, 287 Changhuai Road, Bengbu, China; \u003csup\u003e28\u003c/sup\u003eDepartment of Oncology, Nanshi Hospital of Nanyang, No.130 Zhongzhou West Road, Nanyang 473001, China; \u003csup\u003e29\u003c/sup\u003eDepartment of Oncology, The First Affiliated Hospital of Nanchang University, No.17 Yongwai Main Street, Nanchang 330006, China; \u003csup\u003e30\u003c/sup\u003eWuhan YZY Biopharma Co. Ltd., C2-1 Building, Guanggu Biolake, No. 666, Gaoxin Avenue, Wuhan 430000, China; \u003csup\u003e31\u003c/sup\u003eDepartment of Oncology, The Fifth Medical Center, Chinese PLA General Hospital, No.8 Dongdajie, Beijing 100071, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaif MW, Siddiqui IA, Sohail MA. Management of ascites due to gastrointestinal malignancy. Ann Saudi Med. 2009;29(5):369\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAyantunde AA, Parsons SL. Pattern and prognostic factors in patients with malignant ascites: a retrospective study. Ann Oncol. 2007;18(5):945\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eParsons SL, Watson SA, Steele RJ. Malignant ascites. Br J Surg. 1996;83(1):6\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSangisetty SL, Miner TJ. Malignant ascites: A review of prognostic factors, pathophysiology and therapeutic measures. World J Gastrointest Surg. 2012;4(4):87\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIkegami T, Ishiki H, Kadono T, Ito T, Yokomichi N. Narrative review of malignant ascites: epidemiology, pathophysiology, assessment, and treatment. Ann Palliat Med. 2024;13(4):842\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatsusaki K, Aridome K, Emoto S, Kajiyama H, Takagaki N, Takahashi T, Tsubamoto H, Nagao S, Watanabe A, Shimada H, et al. Clinical practice guideline for the treatment of malignant ascites: section summary in Clinical Practice Guideline for peritoneal dissemination (2021). Int J Clin Oncol. 2022;27(1):1\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStukan M. Drainage of malignant ascites: patient selection and perspectives. Cancer Manag Res. 2017;9:115\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLitvinov SV, Velders MP, Bakker HA, Fleuren GJ, Warnaar SO. Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule. J Cell Biol. 1994;125(2):437\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBalzar M, Briaire-de Bruijn IH, Rees-Bakker HA, Prins FA, Helfrich W, de Leij L, Riethm\u0026uuml;ller G, Alberti S, Warnaar SO, Fleuren GJ, et al. Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions. Mol Cell Biol. 2001;21(7):2570\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBalzar M, Winter MJ, de Boer CJ, Litvinov SV. The biology of the 17-1A antigen (Ep-CAM). J Mol Med (Berl). 1999;77(10):699\u0026ndash;712.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWent P, Vasei M, Bubendorf L, Terracciano L, Tornillo L, Riede U, Kononen J, Simon R, Sauter G, Baeuerle PA. Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers. Br J Cancer. 2006;94(1):128\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWent PT, Lugli A, Meier S, Bundi M, Mirlacher M, Sauter G, Dirnhofer S. Frequent EpCam protein expression in human carcinomas. Hum Pathol. 2004;35(1):122\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu S, Li A, Liu Q, Yuan X, Xu H, Jiao D, Pestell RG, Han X, Wu K. Recent advances of bispecific antibodies in solid tumors. J Hematol Oncol. 2017;10(1):155.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGastl G, Spizzo G, Obrist P, D\u0026uuml;nser M, Mikuz G. Ep-CAM overexpression in breast cancer as a predictor of survival. Lancet. 2000;356(9246):1981\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpizzo G, Obrist P, Ensinger C, Theurl I, D\u0026uuml;nser M, Ramoni A, Gunsilius E, Eibl G, Mikuz G, Gastl G. Prognostic significance of Ep-CAM AND Her-2/neu overexpression in invasive breast cancer. Int J Cancer. 2002;98(6):883\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpizzo G, Went P, Dirnhofer S, Obrist P, Moch H, Baeuerle PA, Mueller-Holzner E, Marth C, Gastl G, Zeimet AG. Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. Gynecol Oncol. 2006;103(2):483\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVarga M, Obrist P, Schneeberger S, M\u0026uuml;hlmann G, Felgel-Farnholz C, Fong D, Zitt M, Brunhuber T, Sch\u0026auml;fer G, Gastl G, et al. Overexpression of epithelial cell adhesion molecule antigen in gallbladder carcinoma is an independent marker for poor survival. Clin Cancer Res. 2004;10(9):3131\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eM\u0026uuml;nz M, Kieu C, Mack B, Schmitt B, Zeidler R, Gires O. The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation. Oncogene. 2004;23(34):5748\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOsta WA, Chen Y, Mikhitarian K, Mitas M, Salem M, Hannun YA, Cole DJ, Gillanders WE. EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res. 2004;64(16):5818\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrinkmann U, Kontermann RE. Bispecific antibodies. Science. 2021;372(6545):916\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLabrijn AF, Janmaat ML, Reichert JM, Parren P. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019;18(8):585\u0026ndash;608.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGoebeler ME, Bargou RC. T cell-engaging therapies - BiTEs and beyond. Nat Rev Clin Oncol. 2020;17(7):418\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan de Donk N, Zweegman S. T-cell-engaging bispecific antibodies in cancer. Lancet. 2023;402(10396):142\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeclercq G, Steinhoff N, Haegel H, De Marco D, Bacac M, Klein C. Novel strategies for the mitigation of cytokine release syndrome induced by T cell engaging therapies with a focus on the use of kinase inhibitors. Oncoimmunology. 2022;11(1):2083479.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeclercq-Cohen G, Steinhoff N, Albert\u0026iacute; Servera L, Nassiri S, Danilin S, Piccione E, Y\u0026aacute;ng\u0026uuml;ez E, H\u0026uuml;sser T, Herter S, Schmeing S, et al. Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies. Clin Cancer Res. 2023;29(21):4449\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMandikian D, Takahashi N, Lo AA, Li J, Eastham-Anderson J, Slaga D, Ho J, Hristopoulos M, Clark R, Totpal K, et al. Relative Target Affinities of T-Cell-Dependent Bispecific Antibodies Determine Biodistribution in a Solid Tumor Mouse Model. Mol Cancer Ther. 2018;17(4):776\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStaflin K, Zuch de Zafra CL, Schutt LK, Clark V, Zhong F, Hristopoulos M, Clark R, Li J, Mathieu M, Chen X et al. Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody. JCI Insight 2020, 5(7).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZuch de Zafra CL, Fajardo F, Zhong W, Bernett MJ, Muchhal US, Moore GL, Stevens J, Case R, Pearson JT, Liu S, et al. Targeting Multiple Myeloma with AMG 424, a Novel Anti-CD38/CD3 Bispecific T-cell-recruiting Antibody Optimized for Cytotoxicity and Cytokine Release. Clin Cancer Res. 2019;25(13):3921\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrinklein ND, Pham D, Schellenberger U, Buelow B, Boudreau A, Choudhry P, Clarke SC, Dang K, Harris KE, Iyer S, et al. Efficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies. MAbs. 2019;11(4):639\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDang K, Castello G, Clarke SC, Li Y, Balasubramani A, Boudreau A, Davison L, Harris KE, Pham D, Sankaran P et al. Attenuating CD3 affinity in a PSMAxCD3 bispecific antibody enables killing of prostate tumor cells with reduced cytokine release. J Immunother Cancer 2021, 9(6).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu CY, Ahonen CL, Brown ME, Zhou L, Welin M, Krauland EM, Pejchal R, Widboom PF, Battles MB. Structure-based engineering of a novel CD3ε-targeting antibody for reduced polyreactivity. MAbs. 2023;15(1):2189974.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePouleau B, Estoppey C, Suere P, Nallet E, Laurendon A, Monney T, Pais Ferreira D, Drake A, Carretero-Iglesia L, Macoin J, et al. Preclinical characterization of ISB 1342, a CD38 \u0026times; CD3 T-cell engager for relapsed/refractory multiple myeloma. Blood. 2023;142(3):260\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSebastian M, Kuemmel A, Schmidt M, Schmittel A. Catumaxomab: a bispecific trifunctional antibody. Drugs Today (Barc). 2009;45(8):589\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeimetz D, Lindhofer H, Bokemeyer C. Development and approval of the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3) as a targeted cancer immunotherapy. Cancer Treat Rev. 2010;36(6):458\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi YN, Li YY, Wang SX, Ma XY. Efficacy of Bispecific Antibody Targeting EpCAM and CD3 for Immunotherapy in Ovarian Cancer Ascites: An Experimental Study. Curr Med Sci. 2023;43(3):539\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu J, Zhao C, Jia Y, Wang S, Ma X, Wang T, Huang S, Pei M, Wang X, Zhou P. 539P Interim results of a phase I study of M701, a recombinant anti-EpCAM and anti-CD3 bispecific antibody in EpCAM-positive cancer patients with malignant ascites. Ann Oncol. 2021;32:S603.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai J, Zhang F, Song Z, Jin J, Lv D, Pang W, Yi T, Wang G, Yao J, Wang B, et al. 1371P An anti-EpCAM x CD3 bispecific antibody, M701, for the treatment of malignant pleural effusion in NSCLC patients: Intermediate results of a prospective multicenter phase Ib trial. Ann Oncol. 2024;35:S862.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeiss MM, Murawa P, Koralewski P, Kutarska E, Kolesnik OO, Ivanchenko VV, Dudnichenko AS, Aleknaviciene B, Razbadauskas A, Gore M, et al. The trifunctional antibody catumaxomab for the treatment of malignant ascites due to epithelial cancer: Results of a prospective randomized phase II/III trial. Int J Cancer. 2010;127(9):2209\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSebastian M, Kiewe P, Schuette W, Brust D, Peschel C, Schneller F, R\u0026uuml;hle KH, Nilius G, Ewert R, Lodziewski S, et al. Treatment of malignant pleural effusion with the trifunctional antibody catumaxomab (Removab) (anti-EpCAM x Anti-CD3): results of a phase 1/2 study. J Immunother. 2009;32(2):195\u0026ndash;202.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMau-S\u0026oslash;rensen M, Dittrich C, Dienstmann R, Lassen U, B\u0026uuml;chler W, Martinius H, Tabernero J. A phase I trial of intravenous catumaxomab: a bispecific monoclonal antibody targeting EpCAM and the T cell coreceptor CD3. Cancer Chemother Pharmacol. 2015;75(5):1065\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJacquet P, Sugarbaker PH. Effects of postoperative intraperitoneal chemotherapy on peritoneal wound healing and adhesion formation. Cancer Treat Res. 1996;82:327\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLotti M, Giulii Capponi M, Campanati L, Poiasina E, Ansaloni L, Poletti E, Frigerio L. The Onset of Intra-Abdominal Adhesions During Closed-Abdomen Hyperthermic Intraperitoneal Chemotherapy. J Laparoendosc Adv Surg Tech A. 2016;26(12):997\u0026ndash;1002.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeiss MM, Strohlein MA, Bokemeyer C, Arnold D, Parsons SL, Seimetx D, et al. The role of relative lymphocyte count as a biomarker for the effect of catumaxomab on survival in malignant ascites patients: results from a phase II/III study. Clin Cancer Res. 2014;20(12):3348\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-hematology-and-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"exho","sideBox":"Learn more about [Experimental Hematology \u0026 Oncology](http://ehoonline.biomedcentral.com)","snPcode":"40164","submissionUrl":"https://submission.nature.com/new-submission/40164/3","title":"Experimental Hematology \u0026 Oncology","twitterHandle":"@SN_Oncology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"EpCAM, CD3, bispecific antibody, malignant ascites, intraperitoneal therapy, puncture-free survival","lastPublishedDoi":"10.21203/rs.3.rs-7204041/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7204041/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMalignant ascites (MA) is one of the major complications in advanced epithelial cancer patients and is associated with poor prognosis, poor quality of life, and severe symptoms. No efficient medicine is available for treating MA worldwide. Only paracentesis is recommended by the guidelines in most countries, but with limited efficacy and a short control time. Thus, novel treatments are needed to control MA.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eAn anti-EpCAM \u0026times; anti-CD3 bispecific antibody, M701, was constructed as a T-cell engager to eliminate tumor cells in the peritoneal cavity. A phase 2 study was performed to evaluate the efficacy and safety of the intraperitoneal (IP) infusion of M701 in advanced epithelial tumor patients with moderate-to-large-scale MA. In this study, 84 patients were enrolled, with 43 in the M701 group receiving paracentesis and IP M701 infusion and 41 in the control group receiving paracentesis alone.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe major endpoint, median puncture-free survival (PuFS), was 75 days in the M701 group and 25 days in the control group, with a significant difference (p\u0026thinsp;=\u0026thinsp;0.0065). Subgroup analysis indicated that different types of cancer, including gastric, colorectal, and ovarian cancers, all benefited from the M701 infusion. Patients with higher relative lymphocyte counts (\u0026ge;\u0026thinsp;13%) at baseline received better effects. Compared to those in the control group, the overall survival (OS) of patients in the M701 group was longer (mOS 110 days vs 76 days, p\u0026thinsp;=\u0026thinsp;0.1443, HR\u0026thinsp;=\u0026thinsp;0.68). The 6-month survival rates were 33.3% and 12.1% in the two groups, respectively. No additional serious adverse events (SAEs) were detected in the M701 group. The most frequent treatment-related adverse events were anemia and low white blood cell count, which were manageable. M701 infusions did not cause a greater risk than paracentesis alone in the control arm, while all patients were administered systemic treatment.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eWhen treated with M701, patients with MA had significantly longer puncture intervals and trend of longer survival times. The results were encouraging for patients with MA. A phase III clinical trial of M701 aimed at further validation is ongoing.\u003c/p\u003e","manuscriptTitle":"Development and Clinical Trial of M701, an Anti-EpCAM × Anti-CD3 Bispecific Antibody: A Targeted Intraperitoneal Therapy for Malignant Ascites Stemming from Advanced Solid Tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-05 12:24:16","doi":"10.21203/rs.3.rs-7204041/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-25T13:09:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-15T14:46:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-07T14:43:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116954588347747714629465915644864908319","date":"2025-08-07T14:03:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-06T22:43:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255618337306190242001697532055350183117","date":"2025-08-06T11:52:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20603787056531875795750759121494332711","date":"2025-08-05T11:11:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252763773063083652780304754613889522460","date":"2025-08-03T05:57:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-30T14:33:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-25T00:34:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-24T18:23:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental Hematology \u0026 Oncology","date":"2025-07-24T09:35:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-hematology-and-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"exho","sideBox":"Learn more about [Experimental Hematology \u0026 Oncology](http://ehoonline.biomedcentral.com)","snPcode":"40164","submissionUrl":"https://submission.nature.com/new-submission/40164/3","title":"Experimental Hematology \u0026 Oncology","twitterHandle":"@SN_Oncology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9bdc35a6-06e3-466e-95a7-2f982a755145","owner":[],"postedDate":"August 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T15:59:43+00:00","versionOfRecord":{"articleIdentity":"rs-7204041","link":"https://doi.org/10.1186/s40164-025-00727-3","journal":{"identity":"experimental-hematology-and-oncology","isVorOnly":false,"title":"Experimental Hematology \u0026 Oncology"},"publishedOn":"2025-11-22 15:56:59","publishedOnDateReadable":"November 22nd, 2025"},"versionCreatedAt":"2025-08-05 12:24:16","video":"","vorDoi":"10.1186/s40164-025-00727-3","vorDoiUrl":"https://doi.org/10.1186/s40164-025-00727-3","workflowStages":[]},"version":"v1","identity":"rs-7204041","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7204041","identity":"rs-7204041","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}