Purpose: Near-infrared fluorescence-guided surgery (FGS) using cancer-specific tracers is promising for tailored gastric cancer (GC) surgery. Carcinoembryonic antigen (CEA) is a potential target due to its high expression in various digestive cancers, including GC. Materials and Methods: SGM-101, a chimeric anti-CEA monoclonal antibody conjugated with the near-infrared dye BM-104, was evaluated in GC. CEA expression was identified in GC cell lines at the mRNA and protein levels. Xenograft models (MKN-45, SNU-16, SNU-668, 85As2mLuc) were established in mice and injected with SGM-101 or PBS. Biodistribution was monitored using in vivo fluorescence imaging. Tumors were further analyzed by immunofluorescence. In a peritoneal carcinomatosis model, 85As2mLuc cells were injected intraperitoneally, and tumors were evaluated by bioluminescence and fluorescence and histology. Results: MKN-45, SNU-16, and 85As2mLuc were CEA-positive, while SNU-668 was CEA-negative. Flow cytometry confirmed CEA expression: MKN-45 (98%), SNU-16 (85.6%), SNU-668 (6.42%) and 85As2mLuc (78.4%). SGM-101 selectively targeted CEA-expressing tumors, with fluorescence peaking at 48 h, and immunofluorescence verified localization in tumor cells. In the peritoneal models, SGM-101 enabled precise detection of CEA-positive tumors. Conclusions: This study provides the first evidence for the feasibility of SGM-101 in gastric cancer, demonstrating its novelty and translational potential as a cancer-specific imaging agent for fluorescence-guided surgery.

Progastrin inhibition decreases colonosphere incidence, CSC frequency and ALDHhigh cell proportion in vitro.

Supplementary Legends for Figures 1-3, Videos 1-2 from R-Cadherin Expression Inhibits Myogenesis and Induces Myoblast Transformation via Rac1 GTPase

Description of additional methods and procedures used in the study.

BACKGROUND Intraoperative molecular imaging has emerged as a potential tool in addressing challenges faced during lung cancer surgery by localizing small lesions, ensuring negative margins, and identifying synchronous cancers. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) glycoprotein has emerged as a potential target in fluorescent labeling of non-small cell lung cancer given the high antigen density in tumor cells and absence of expression in normal parenchyma. The goal of our study was to determine whether anti-CEACAM5 targeted near-infrared fluorochrome could be a suitable target in non-small cell lung cancer.METHODS The CEACAM5 expression was evaluated in AB-12 (known negative control), HT29 (known positive control), and H460 (non-small cell lung cancer) cell lines by polymerase chain reaction. SGM-101, a CEACAM5 antibody, coupled with a BM-104 near-infrared fluorescent tracer was evaluated with dose escalation, in vitro cellular localization, and immunofluorescence microscopy. Subsequently, in vivo validation was performed in 52 athymic nude xenografts.RESULTS Polymerase chain reaction analysis demonstrated 3000x relative expression of CEACAM5 in HT-29 cells compared with AB-12. The H460 cells showed 1000x relative expression compared with AB12 (P < .05). Both HT29 and H460 cells showed tracer internalization with signal to background ratio of 4.5 (SD 0.34) whereas there was minimal uptake by AB12 cells with signal to background ratio 1.1 (SD 0.1; P < .05). There was linear fluorescence increase with increasing tracer dosing in receptor expressing cell lines. In preclinical models, HT-29 and H460 cells lines produced near-infrared fluorescence with average tumor to background ratio of 3.89 (SD 0.25) irrespective of tumor size compared with no fluorescence by AB12 tumors (P < .05). The CEACAM5 expressing tumors had excellent dye uptake compared with AB12 tumors.CONCLUSIONS CEACAM5 serves as a possible receptor for targeted intraoperative molecular imaging resections in lung cancer. This study sets a path for evaluation of CEACAM5 targets in future clinical trials.(c) 2023 by The Society of Thoracic Surgeons.

shRNA-mediated down regulation of progastrin in vitro and in vivo.

Figure S3: Characterization of UT-7/Epo, HMC1.1 and HMC1.2 cell lines. Figure S4: 2D1 and 3G1-Fc do not compete with 104D2 mAb binding to KIT.

Supplemental Fig.S3. In pancreatic cancer cells, ch9F7-F11-Emb is a negative allosteric modulator of NRG1-mediated signaling. The low-fucose non-NRG1 competing allosteric anti-HER3 antibody ch9F7-F11-Emb inhibits in a dose-dependent manner NRG1-mediated cell signaling more efficiently than the ligand-competing H4B-121-Emb antibody. BxPC3 cells were pre-stimulated with 1nM NRG1 for 5min, before adding antibodies at various concentrations for another 25min. After cell lysis, the expression levels of total and phosphorylated HER3 (Tyr1289, Tyr1197 and Tyr1222), and total and phosphorylated AKT (Ser473) were measured by western blotting (A) using the appropriate antibodies. Phosphorylation was then pixel-quantified with Image J (B) and shown relative to the maximal phosphorylation (100%) evaluated in NRG1-stimulated cells without antibody treatment (M). Results are the mean {plus minus} SD of three independent experiments

Importance:Localization of subcentimeter ground glass opacities during minimally invasive thoracoscopic lung cancer resections is a significant challenge in thoracic oncology. Intraoperative molecular imaging has emerged as a potential solution, but the availability of suitable fluorescence agents is a limiting factor.Objective:To evaluate the suitability of SGM-101, a carcinoembryonic antigen-related cell adhesion molecule type 5 (CEACAM5) receptor-targeted near-infrared fluorochrome, for molecular imaging-guided lung cancer resections, because glycoprotein is expressed in more than 80% of adenocarcinomas.Design, Setting, and Participants:For this nonrandomized, proof-of-principal, phase 1 controlled trial, patients were divided into 2 groups between August 1, 2020, and January 31, 2022. Patients with known CEACAM5-positive gastrointestinal tumors suggestive of lung metastasis were selected as proof-of-principle positive controls. The investigative group included patients with lung nodules suggestive of primary lung malignant neoplasms. Patients 18 years or older without significant comorbidities that precluded surgical exploration with suspicious pulmonary nodules requiring surgical biopsy were included in the study.Interventions:SGM-101 (10 mg) was infused up to 5 days before index operation, and pulmonary nodules were imaged using a near-infrared camera system with a dedicated thoracoscope.Main Outcomes and Measures:SGM-101 localization to pulmonary nodules and its correlation with CEACAM5 glycoprotein expression by the tumor as quantified by tumor and normal pulmonary parenchymal fluorescence.Results:Ten patients (5 per group; 5 male and 5 female; median [IQR] age, 66 [58-69] years) with 14 total lesions (median [range] lesion size, 0.91 [0.90-2.00] cm) were enrolled in the study. In the control group of 4 patients (1 patient did not undergo surgical resection because of abnormal preoperative cardiac clearance findings that were not deemed related to SGM-101 infusion), the mean (SD) lesion size was 1.33 (0.48) cm, 2 patients had elevated serum CEA markers, and 2 patients had normal serum CEA levels. Of the 4 patients who underwent surgical intervention, those with 2+ and 3+ tissue CEACAM5 expression had excellent tumor fluorescence, with a mean (SD) tumor to background ratio of 3.11 (0.45). In the patient cohort, the mean (SD) lesion size was 0.68 (0.22) cm, and no elevations in serum CEA levels were found. Lack of SGM-101 fluorescence was associated with benign lesions and with lack of CEACAM5 staining.Conclusions and Relevance:This in-human proof-of-principle nonrandomized controlled trial demonstrated SGM-101 localization to CEACAM5-positive tumors with the detection of real-time near-infrared fluorescence in situ, ex vivo, and by immunofluorescence microscopy. These findings suggest that SGM-101 is a safe, receptor-specific, and feasible intraoperative molecular imaging fluorochrome that should be further evaluated in randomized clinical trials.Trial Registration:ClinicalTrials.gov identifier: NCT04315467.

Biodistribution in mice with subcutaneous LS174T tumors at 24, 48 and 72 hours after injection of 30 μg of [111In]In-DTPA-SGM-101.

Supplemental Fig.S1. NRG1 is a positive-allosteric modulator for 9F7-F11/HER3 binding. Differently from H4B-121 and MAB3481, 9F7-F11 does not compete with the ligand NRG1, and 9F7-F11 binding to HER3 increases in the presence of NRG1. SNAP-Tag Lumi4TM-Tb HER3-expressing HEK cells were co-incubated with various concentrations of unlabeled NRG1 and 0.5nM d2-conjugated antibodies. After 5h30 incubation, the time-resolved fluorescence (665 nm/620 nm emission delta ratio) of the binding of d2-labeled antibodies to HER3 was measured on a Pherastar reader and normalized to 100% binding (antibody-d2 conjugates without NRG1, or NRG1-d2 conjugates without antibodies). Data were plotted using the GraphPad Prism software.

Supplemental Fig.S6. Effect of NRG1 on cell viability of the cancer cell lines BxPC3, HCC-1806, A549 and HPAC. Cells were plated in medium with 1% fetal calf serum and then cultured with increasing concentrations of NRG1 for 5 days. Cell proliferation was measured using the MTS assay.

Annexin II expression is not enriched in cancer stem cell populations.

Progastrin regulates the self-renewal of ALDHhigh colorectal CSCs.

Suppl.Table 1: Analysis of the cellular localization, pathways and gene expression modification of the 100 genes most correlated with AXL expression in 254 basal-like breast cancers.

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations. We then screened these bispecific antibodies and compared them with the parental single antibodies and antibody pair combinations. The screen readouts included measuring binding to the cognate receptors (mono and bispecificity), intracellular phosphorylation signaling, cell proliferation, apoptosis and receptor expression, and also immune system engagement assays (antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity). Among the 30 BiXAbs™ tested, we selected 3Patri-1Cetu-Fc, 3Patri-1Matu-Fc and 3Patri-2Trastu-Fc as lead candidates. The in vivo testing of these three highly efficient bispecific antibodies against EGFR and HER2 or HER3 in pre-clinical mouse models of pancreatic cancer showed deep antibody penetration in these dense tumors and robust tumor growth reduction. Application of such semi-rational/semi-empirical approach, which includes various immunological assays to compare pre-selected antibodies and their combinations with bispecific antibodies, represents the first attempt to identify potent bispecific antibodies against ErbB family members in pancreatic cancer.

Monitoring of phage antibody selection by titration, ELISA and scFv sequencing.

ALDHhigh CRC cells display CSC characteristics and elevated progastrin expression.

Microscopic analysis of resected intraperitoneal tumors shows clear overlap of CEA-expressing tumor cells, NIR-fluorescence and radiosignal. In the bottom right corner a region with normal non-CEA expressing cells clearly shows low fluorescence and radiosignal. (A) H&E, B) CEA, C) NIR-Fluorescence, D) Autoradiography.

Materials and methods describing the production of scFv-Fc in HEK-T cells and KIT domains and scFv display on yeast. Table S2: Primers for PCR amplification of KIT and cloning into pYD vector by Gap repair. Figure S1: Selection and initial characterization of a panel of anti-KIT scFv. Figure S2: Anti-KIT D5 epitope characterization using yeast display.