EpCAM Signaling Promotes Tumor Progression and Protein Stability of PD-L1 through the EGFR Pathway.

Although epithelial cell adhesion molecule (EpCAM) has previously been shown to promote tumor progression, the underlying mechanisms remain largely unknown. Here, we report that the EGF-like domain I within the extracellular domain of EpCAM (EpEX) binds EGFR, activating both AKT and MAPK signaling to inhibit forkhead transcription factor O3a (FOXO3a) function and stabilize PD-L1 protein, respectively. Treatment with the EpCAM neutralizing antibody, EpAb2-6, inhibited AKT and FOXO3a phosphorylation, increased FOXO3a nuclear translocation, and upregulated high temperature requirement A2 (HtrA2) expression to promote apoptosis while decreasing PD-L1 protein levels to enhance the cytotoxic activity of CD8+ T cells. In vivo, EpAb2-6 markedly extended survival in mouse metastasis and orthotopic models of human colorectal cancer. The combination of EpAb2-6 with atezolizumab, an anti-PD-L1 antibody, almost completely eliminated tumors. Moreover, the number of CD8+ T cells in combination-treated tumors was increased compared with atezolizumab alone. Our findings suggest a new combination strategy for cancer immunotherapy in patients with EpCAM-expressing tumors. SIGNIFICANCE: This study shows that treatment with an EpCAM neutralizing antibody promotes apoptosis while decreasing PD-L1 protein to enhance cytotoxic activity of CD8+ T cells.

Epitope resurfacing on dengue virus-like particle vaccine preparation to induce broad neutralizing antibody.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of 'epitope-resurfaced' mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.

An anti-EpCAM antibody EpAb2-6 for the treatment of colon cancer.

Epithelial cell adhesion molecule (EpCAM) is known to be overexpressed in epithelial cancers associated with enhanced malignant potential, particularly colorectal carcinoma (CRC) and head and neck squamous cell carcinoma (HNSCC). However, it is unknown whether progression of malignance can be directly inhibited by targeting EpCAM. Here, we have generated five novel monoclonal antibodies (mAbs) against EpCAM. One of these anti-EpCAM mAbs, EpAb2-6, was found to induce cancer cell apoptosis in vitro, inhibit tumor growth, and prolong the overall survival of both a pancreatic cancer metastatic mouse model and mice with human colon carcinoma xenografts. EpAb2-6 also increases the therapeutic efficacy of irinotecan, fluorouracil, and leucovorin (IFL) therapy in a colon cancer animal model and gemcitabine therapy in a pancreatic cancer animal model. Furthermore, EpAb2-6, which binds to positions Y95 and D96 of the EGF-II/TY domain of EpCAM, inhibits production of EpICD, thereby decreasing its translocation and subsequent signal activation. Collectively, our results indicate that the novel anti-EpCAM mAb can potentially be used for cancer-targeted therapy.

Generation of Monoclonal Antibodies against Dengue Virus Type 4 and Identification of Enhancing Epitopes on Envelope Protein.

The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.

An Epitope-Substituted DNA Vaccine Improves Safety and Immunogenicity against Dengue Virus Type 2.

Dengue virus (DENV), a global disease, is divided into four serotypes (DENV1-4). Cross-reactive and non-neutralizing antibodies against envelope (E) protein of DENV bind to the Fcγ receptors (FcγR) of cells, and thereby exacerbate viral infection by heterologous serotypes via antibody-dependent enhancement (ADE). Identification and modification of enhancing epitopes may mitigate enhancement of DENV infection. In this study, we characterized the cross-reactive DB21-6 and DB39-2 monoclonal antibodies (mAbs) against domain I-II of DENV; these antibodies poorly neutralized and potently enhanced DENV infection both in vitro and in vivo. In addition, two enhancing mAbs, DB21-6 and DB39-2, were observed to compete with sera antibodies from patients infected with dengue. The epitopes of these enhancing mAbs were identified using phage display, structural prediction, and mapping of virus-like particle (VLP) mutants. N8, R9, V12, and E13 are the reactive residues of DB21-6, while N8, R9, and E13 are the reactive residues of DB39-2. N8 substitution tends to maintain VLP secretion, and decreases the binding activity of DB21-6 and DB39-2. The immunized sera from N8 substitution (N8R) DNA vaccine exerted greater neutralizing and protective activity than wild-type (WT)-immunized sera, both in vitro and in vivo. Furthermore, treatment with N8R-immunized sera reduced the enhancement of mortality in AG129 mice. These results support identification and substitution of enhancing epitope as a novel strategy for developing safe dengue vaccines.

Generation of an anti-EpCAM antibody and epigenetic regulation of EpCAM in colorectal cancer.

We have generated a novel monoclonal antibody (mAb), OCAb9-1, which specifically binds to various types of cancer cell lines, but not to normal cells. According to the results of immunoaffinity chromatography, LC-MS/MS analysis, co-immunoprecipitation, and RNA interference studies, the target protein of OCAb9-1 is the epithelial cell adhesion molecule (EpCAM). EpCAM is a type I transmembrane glycoprotein which is highly expressed in epithelial-transformed neoplasia and tumor-initiating cells (TICs). However, regulation of EpCAM gene expression in tumors and its role in tumorigenesis are not fully understood. In the present study, we show that EpCAM expression is elevated in several cancer cell lines and tumor tissues. Loss-of-function experiments were performed to demonstrate that EpCAM negatively regulates expression of p53 and p21, and promotes tumor cell growth, colony formation, migration and invasion. The median overall survival of tumor-bearing mice treated with OCAb9-1 was significantly higher than that of PBS-treated mice. Moreover, we report that the interplay between SUZ12 and JMJD3 results in dynamic regulation of lysine 27 trimethylation of histone 3 (H3K27me3). Taken together, our findings suggest that the anti-EpCAM mAb may be suitable for use in cancer diagnosis, prognosis, imaging and therapy. Furthermore, EpCAM overexpression in cancer cells is strongly associated with tumor progression, and may be regulated by epigenetic mechanisms.

Podocalyxin-like 1 promotes invadopodia formation and metastasis through activation of Rac1/Cdc42/cortactin signaling in breast cancer cells.

Metastatic disease is the leading cause of cancer mortality. Identifying biomarkers and regulatory mechanisms is important toward developing diagnostic and therapeutic tools against metastatic cancer. In this study, we demonstrated that podocalyxin-like 1 (PODXL) is overexpressed in breast tumor cells and increased in lymph node metastatic cancer. Mechanistically, we found that the expression of PODXL was associated with cell motility and invasiveness. Suppression of PODXL in MDA-MB-231 cells reduced lamellipodia formation and focal adhesion kinase (FAK) and paxillin phosphorylation. PODXL knockdown reduced the formation of invadopodia, such as inhibiting the colocalization of F-actin with cortactin and suppressing phosphorylation of cortactin and neural Wiskott-Aldrich syndrome protein. Conversely, overexpression of PODXL in MCF7 cells induced F-actin/cortactin colocalization and enhanced invadopodia formation and activation. Invadopodia activity and tumor invasion in PODXL-knockdown cells are similar to that in cortactin-knockdown cells. We further found that the DTHL motif in PODXL is crucial for regulating cortactin phosphorylation and Rac1/Cdc42 activation. Inhibition of Rac1/Cdc42 impeded PODXL-mediated cortactin activation and FAK and paxillin phosphorylation. Moreover, inhibition of PODXL in MDA-MB-231 cells significantly suppressed tumor colonization in the lungs and distant metastases, similar to those in cortactin-knockdown cells. These findings show that overexpression of PODXL enhanced invadopodia formation and tumor metastasis by inducing Rac1/Cdc42/cortactin signaling network.

Antibody conjugated supported lipid bilayer for capturing and purification of viable tumor cells in blood for subsequent cell culture.

Interest in the identification and isolation of circulating tumor cells (CTCs) has been growing since the introduction of CTCs as an alternative to the tumor tissue biopsy, which can potentially be important indices for prognosis and cancer treatment. However, the contamination of non-specific binding of normal hematologic cells makes high purity CTCs detection problematic. Furthermore, preserving the viability of CTCs remains a challenge. In this study, we proposed to construct an anti-EpCAM functionalized supported lipid bilayer (SLB), a biomimetic and non-fouling membrane coating, for CTCs capturing, purification and maintaining the viability. Healthy human blood spiked with pre-stained colorectal cancer cell lines, HCT116 and colo205, were used to investigate interaction of cells with the anti-EpCAM functionalized SLB surfaces. Over 97% of HCT116, and 72% of colo205 were captured and adhered by the surface anti-EpCAM; conversely, the majority of blood cells were easily removed by gentle buffer exchange, with the overall purity of cancer cells exceeding 95%. The bound cancer cells were subsequently detached for cell culture. Both HCT116 and colo205 continued to proliferate over 2-week observation period, indicating that the anti-EpCAM functionalized SLB platform providing a simple strategy for capturing, purifying, and releasing viable targeted rare cells.

Analysis of epitopes on dengue virus envelope protein recognized by monoclonal antibodies and polyclonal human sera by a high throughput assay.

BACKGROUND: The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC' loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. CONCLUSIONS/SIGNIFICANCE: Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.

Epithelial cell adhesion molecule regulation is associated with the maintenance of the undifferentiated phenotype of human embryonic stem cells.

Human embryonic stem cells (hESCs) are unique pluripotent cells capable of self-renewal and differentiation into all three germ layers. To date, more cell surface markers capable of reliably identifying hESCs are needed. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein expressed in several progenitor cell populations and cancers. It has been used to enrich cells with tumor-initiating activity in xenograft transplantation studies. Here, we comprehensively profile the expression of EpCAM by immunofluorescence microscopy, Western blotting, and flow cytometry using an anti-EpCAM monoclonal antibody (mAb) OC98-1. We found EpCAM to be highly and selectively expressed by undifferentiated rather than differentiated hESCs. The protein and transcript level of EpCAM rapidly diminished as soon as hESC had differentiated. This silencing was closely and exclusively associated with the radical transformation of histone modification at the EpCAM promoter. Moreover, we demonstrated that the dynamic pattern of lysine 27 trimethylation of histone 3 was conferred by the interplay of SUZ12 and JMJD3, both of which were involved in maintaining hESC pluripotency. In addition, we used chromatin immunoprecipitation analysis to elucidate the direct regulation by EpCAM of several reprogramming genes, including c-MYC, OCT-4, NANOG, SOX2, and KLF4, to help maintain the undifferentiation of hESCs. Collectively, our results suggest that EpCAM might be used as a surface marker for hESC. The expression of EpCAM may be regulated by epigenetic mechanisms, and it is strongly associated with the maintenance of the undifferentiated state of hESCs.

Disease-specific B Cell epitopes for serum antibodies from patients with severe acute respiratory syndrome (SARS) and serologic detection of SARS antibodies by epitope-based peptide antigens.

Severe acute respiratory syndrome (SARS) has emerged as a highly contagious, sometimes fatal disease. To find disease-specific B cell epitopes, phage-displayed random peptide libraries were panned on serum immunoglobulin (Ig) G antibodies from patients with SARS. Forty-nine immunopositive phage clones that bound specifically to serum from patients with SARS were selected. These phageborne peptides had 4 consensus motifs, of which 2 corresponded to amino acid sequences reported for SARS-associated coronavirus (SARS-CoV). Synthetic peptide binding and competitive-inhibition assays further confirmed that patients with SARS generated antibodies against SARS-CoV. Immunopositive phage clones and epitope-based peptide antigens demonstrated clinical diagnostic potential by reacting with serum from patients with SARS. Antibody-response kinetics were evaluated in 4 patients with SARS, and production of IgM, IgG, and IgA were documented as part of the immune response. In conclusion, B cell epitopes of SARS corresponded to novel coronavirus. Our epitope-based serologic test may be useful in laboratory detection of the virus and in further study of the pathogenesis of SARS.