274 CD47 x EpCAM bispecific antibody represents a novel approach for treating EpCAM overexpressing solid tumors

BackgroundCD47 conveys a ”don’t eat me” signal through the interaction with its ligand signal regulatory protein-alpha (SIRPa) on myeloid cells and blocks macrophage mediated phagocytosis. Tumor cells, which express high level of CD47, exploit this mechanism to evade from immune surveillance. CD47/SIRPa axis is an important checkpoint of innate immune system and CD47 is considered a prominent target for cancer treatment.1 However, the wide expression of CD47 on normal cells could cause antigen sink and lead to safety issues, such as anemia and thrombocytopenia. EpCAM is highly expressed in many epithelial cancers, particularly in colorectal, gastric, endometrial and lung cancers. Here we describe a novel CD47 x EpCAM bispecific antibody, which specifically targets CD47+/EpCAM+ tumors in preclinical studies.MethodsThe CD47 x EpCAM bispecific antibody was generated using novel anti-CD47 antibody and anti-EpCAM antibody. A series of in vitro assays including FACS binding, FACS-based SIRPa blocking, ADCP, RBC binding and hemagglutination were performed to characterize the CD47 x EpCAM bispecific antibody. In vivo efficacies of this bispecific antibody were evaluated in xenograft tumor models with high EpCAM and CD47 expression.ResultsCompared to monoclonal CD47 antibody, the CD47 x EpCAM bispecific antibody selectively binds to tumor cells overexpressing EpCAM. The bispecific antibody exhibited potent SIRPa blocking and antibody-dependent cellular phagocytosis (ADCP) activity on CD47+/EpCAM+ tumor cells, but not on cells lacking EpCAM expression. Compared to its parental CD47 monoclonal antibody, the EC50 of SIRPa blocking activity were improved 30–80 folds with the treatment of the CD47 x EpCAM bispecific antibody in tumor cell lines with high EpCAM expression. No significant RBC binding and RBC phagocytosis were observed upon treatment with the CD47 x EpCAM bispecific antibody. The bispecific antibody did not cause any appreciable hemagglutination with up to 1µM antibody treatment. Most importantly, the bispecific antibody demonstrated potent anti-tumor activities in in vivo solid tumor cell line-derived xenograft (CDX) models that overexpress both CD47 and EpCAM.ConclusionsOur findings suggest that the novel CD47 x EpCAM bispecific antibody selectively binds to CD47 and blocks CD47/SIRPa binding on EpCAM overexpressing tumor cells. The bispecific antibody has minimum RBC binding compared to the bivalent CD47 monoclonal antibodies. The bispecific antibody shows potent in vivo efficacy and specificity toward EpCAM positive tumor cells and represents a novel approach for treating EpCAM+ tumors.ReferencesChao M, Weissman I, Majeti R. The CD47-SIRPa pathway in cancer immune evasion and potential therapeutic implications. Curr Opin Immunol 2012;24(2):225–232.Ethics ApprovalThe protocol and any amendment(s) or procedures involving the care and use of animals in these animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec prior to conduct. During the studies, the care and use of animals were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)

Preclinical Evaluation of [99mTc]Tc-Labeled Anti-EpCAM Nanobody for EpCAM Receptor Expression Imaging By Immuno-SPECT/CT

Purpose Overexpression of epithelial cell adhesion molecule (EpCAM) plays essential roles in tumorigenesis and tumor progression in almost all epithelium-derived cancer. Monitoring EpCAM expression in tumors can be used for the diagnosis, staging and prognosis of cancer patients, as well as guiding the individualized treatment of EpCAM-targeted drugs. In this study, we described the synthesis and evaluation of a site-specifically [99mTc]Tc-labeled EpCAM-targeted nanobody for the SPECT/CT imaging of EpCAM expression. Methods We first prepared the [99mTc]Tc-HYNIC-G4K, then it was site-specifically connected to EpCAM-targeted nanobody NB4. The in vitro characteristics of [99mTc]Tc-NB4 were investigated in HT-29 (EpCAM-positive) and HL-60 (EpCAM-negative) cells, while the in vivo studies were performed using small-animal SPECT/CT in the subcutaneous tumor models and the lymph node metastasis model to verify the specific targeting capacity as well as the potential applications of [99mTc]Tc-NB4. Results [99mTc]Tc-NB4 displayed a high EpCAM specificity both in vitro and in vivo. SPECT/CT imaging revealed that [99mTc]Tc-NB4 was cleared rapidly from the blood and normal organs except for the kidneys, and HT-29 tumors were clearly visualized in contrast with HL-60 tumors. The uptake value of [99mTc]Tc-NB4 in HT-29 tumors was increased continuously from 3.77 ± 0.39 %ID/g at 0.5 h to 5.53 ± 0.82 %ID/g at 12 h after injection. Moreover, the [99mTc]Tc-NB4 SPECT/CT could clearly image tumor-infiltrating lymph nodes. Conclusion [99mTc]Tc-NB4 is a broad-spectrum, specific and sensitive SPECT radiotracer for the noninvasive imaging of EpCAM expression in the epithelium-derived cancer, and revealed a great potential for the clinical translation.

Functional Implications of the Dynamic Regulation of EpCAM during Epithelial-to-Mesenchymal Transition

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein expressed in epithelial tissues. EpCAM forms intercellular, homophilic adhesions, modulates epithelial junctional protein complex formation, and promotes epithelial tissue homeostasis. EpCAM is a target of molecular therapies and plays a prominent role in tumor biology. In this review, we focus on the dynamic regulation of EpCAM expression during epithelial-to-mesenchymal transition (EMT) and the functional implications of EpCAM expression on the regulation of EMT. EpCAM is frequently and highly expressed in epithelial cancers, while silenced in mesenchymal cancers. During EMT, EpCAM expression is downregulated by extracellular signal-regulated kinases (ERK) and EMT transcription factors, as well as by regulated intramembrane proteolysis (RIP). The functional impact of EpCAM expression on tumor biology is frequently dependent on the cancer type and predominant oncogenic signaling pathways, suggesting that the role of EpCAM in tumor biology and EMT is multifunctional. Membrane EpCAM is cleaved in cancers and its intracellular domain (EpICD) is transported into the nucleus and binds β-catenin, FHL2, and LEF1. This stimulates gene transcription that promotes growth, cancer stem cell properties, and EMT. EpCAM is also regulated by epidermal growth factor receptor (EGFR) signaling and the EpCAM ectoderm (EpEX) is an EGFR ligand that affects EMT. EpCAM is expressed on circulating tumor and cancer stem cells undergoing EMT and modulates metastases and cancer treatment responses. Future research exploring EpCAM’s role in EMT may reveal additional therapeutic opportunities.

Absence of EpCAM in cervical cancer cells is involved in slug induced epithelial-mesenchymal transition

Background Slug (Snai2) is a pivotal player in initiating epithelial-mesenchymal transition (EMT) through its trans-suppression effect on E-cadherin in various normal and malignant cells. In this study, the positive effect of Slug on promoting cell motility and metastasis in cervical cancer was further confirmed in this study. Methods RNA-Seq was performed to explore the potential molecules that participate in Slug-mediated EMT in cervical cancer cells. The negative correlation between Slug and EpCAM expression in cervical cancer cells was detected in this study, and linked them with in vitro migration and invasion assay, in vivo metastasis experiments, luciferase reporter assay and Chromatin immunoprecipitation. Results Transcriptome sequencing analysis revealed that epithelial cell adhesion molecule (EpCAM) was significantly decreased in Slug-overexpressing SiHa cells. Simultaneously, an absence of EpCAM expression was observed in Slug-overexpressing cells. Further studies revealed the trans-suppression effect of Slug on EpCAM through its binding to the E-boxes in the proximal promoter region of EpCAM in cervical cancer cells. Restoring EpCAM in Slug-overexpressing cells by transiently transfecting an EpCAM recombinant plasmid attenuated cell motility and promoted cell growth. Moreover, the negative correlation between Slug and EpCAM expression in human squamous cervical carcinoma (SCC) samples was verified by using Pearson correlation analysis. Conclusions These results demonstrated that the absence of EpCAM under Slug expression in cervical cancer cells probably participated in Slug-regulated EMT and further promoted tumor metastasis. Additionally, this study supports a potential way for Slug to initiate EMT progression in cervical cancer cells in addition to inhibiting E-cadherin.

Detection of Epithelial Cell Adhesion Molecule in Feline Normal and Tumor Cell Lines and Tissues With Selected Commercial Anti-human EpCAM Antibodies

Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein expressed at intercellular junctions in epithelial cells. As an epithelial biomarker, it used for immunologic-based capture of epithelial-derived circulating tumor cells (CTCs) in human patients with different carcinomas. EpCAM expression has not been described in normal or neoplastic epithelial tissues in cats. Our goal was to find a commercial antibody that recognizes surface EpCAM expression for CTC detection. We tested two anti-human EpCAM antibodies, designated for use with flow cytometry, for detection of surface EpCAM expression on feline cell lines derived from normal mammary and renal epithelia and mammary and oropharyngeal squamous cell carcinomas in cats. Only one of the antibodies, a goat polyclonal antibody, labeled normal and neoplastic feline mammary epithelial cells and oropharyngeal squamous cell carcinoma cells; no labeling was observed for normal feline kidney epithelial cells. At low dilution, this antibody immunohistochemically stained the intercellular junctions of normal pancreatic, intestinal and mammary epithelium, as well as neoplastic mammary epithelium in feline tissues; however, oral mucosa, skin, and an oropharyngeal squamous cell carcinoma showed no positive immunostaining. The antibody only weakly bound feline squamous cell carcinoma cell lines under static adhesion. Our results indicate that EpCAM is expressed in specific epithelia in cats but is variably expressed in feline mammary tumors and oropharyngeal squamous cell carcinoma. A higher avidity cross-reactive or feline-specific antibody will be required to further investigate EpCAM expression in normal and neoplastic feline tissue or for detecting CTCs in the blood of tumor-bearing cats.

EpCAM-Mediated Cellular Plasticity Promotes Radiation Resistance and Metastasis in Breast Cancer

Substantial number of breast cancer (BC) patients undergoing radiation therapy (RT) develop local recurrence over time. During RT therapy, cells can gradually acquire resistance implying adaptive radioresistance. Here we probe the mechanisms underlying this acquired resistance by first establishing radioresistant lines using ZR-75-1 and MCF-7 BC cells through repeated exposure to sub-lethal fractionated dose of 2Gy up to 15 fractions. Radioresistance was found to be associated with increased cancer stem cells (CSCs), and elevated EpCAM expression in the cell population. A retrospective analysis of TCGA dataset indicated positive correlation of high EpCAM expression with poor response to RT. Intriguingly, elevated EpCAM expression in the radioresistant CSCs raise the bigger question of how this biomarker expression contributes during radiation treatment in BC. Thereafter, we establish EpCAM overexpressing ZR-75-1 cells (ZR-75-1EpCAM), which conferred radioresistance, increased stemness through enhanced AKT activation and induced a hybrid epithelial/mesenchymal phenotype with enhanced contractility and invasiveness. In line with these observations, orthotopic implantation of ZR-75-1EpCAM cells exhibited faster growth, lesser sensitivity to radiation therapy and increased lung metastasis than baseline ZR-75-1 cells in mice. In summary, this study shows that similar to radioresistant BC cells, EpCAM overexpressing cells show high degree of plasticity and heterogeneity which ultimately induces radioresistant and metastatic behavior of cancer cells, thus aggravating the disease condition.

GAS2L1 Is a Potential Biomarker of Circulating Tumor Cells in Pancreatic Cancer

Pancreatic cancer is a malignant disease with high mortality and a dismal prognosis. Circulating tumor cell (CTC) detection and characterization have emerged as essential techniques for early detection, prognostication, and liquid biopsy in many solid malignancies. Unfortunately, due to the low EPCAM expression in pancreatic cancer CTCs, no specific marker is available to identify and isolate this rare cell population. This study analyzed single-cell RNA sequencing profiles of pancreatic CTCs from a genetically engineered mouse model (GEMM) and pancreatic cancer patients. Through dimensionality reduction analysis, murine pancreatic CTCs were grouped into three clusters with different biological functions. CLIC4 and GAS2L1 were shown to be overexpressed in pancreatic CTCs in comparison with peripheral blood mononuclear cells (PBMCs). Further analyses of PBMCs and RNA-sequencing datasets of enriched pancreatic CTCs were used to validate the overexpression of GAS2L1 in pancreatic CTCs. A combinatorial approach using both GAS2L1 and EPCAM expression leads to an increased detection rate of CTCs in PDAC in both GEMM and patient samples. GAS2L1 is thus proposed as a novel biomarker of pancreatic cancer CTCs.

Absence of EpCAM in Cervical Cancer Cells Is Involved in Slug-induced Epithelial-mesenchymal Transition

Background: Slug (Snai2) is a pivotal player in initiating epithelial-mesenchymal transition (EMT) through its trans-suppression effect on E-cadherin in various normal and malignant cells. In this study. The positive effect of Slug on promoting cell motility and metastasis in cervical cancer was further confirmed in this study.Methods: RNA-Seq was performed to explore the potential molecules that participate in Slug-mediated EMT in cervical cancer cells. The negative correlation between Slug and EpCAM expression in cervical cancer cells was detected in this study, and linked them with in vitro migration and invasion assay, in vivo metastasis experiments, luciferase reporter assay and Chromatin immunoprecipitation.Results: Transcriptome sequencing analysis revealed that epithelial cell adhesion molecule (EpCAM) was significantly decreased in Slug-overexpressing SiHa cells. Simultaneously, an absence of EpCAM expression was observed in Slug-overexpressing cells. Further studies revealed the trans-suppression effect of Slug on EpCAM through its binding to the E-boxes in the proximal promoter region of EpCAM in cervical cancer cells. Restoring EpCAM in Slug-overexpressing cells by transiently transfecting an EpCAM recombinant plasmid attenuated cell motility and promoted cell growth. Moreover, the negative correlation between Slug and EpCAM expression in human squamous cervical carcinoma (SCC) samples was verified by using Pearson correlation analysis.Conclusions: These results demonstrated that the absence of EpCAM under Slug expression in cervical cancer cells probably participated in Slug-regulated EMT and further promoted tumor metastasis. Additionally, this study supports a potential way for Slug to initiate EMT progression in cervical cancer cells in addition to inhibiting E-cadherin.