A gB/CD3 bispecific BiTE antibody construct for targeting Human Cytomegalovirus-infected cells.

Bispecific T cell engager (BiTE) antibody constructs are successfully used as cancer therapeutics. We hypothesized that this treatment strategy could also be applicable for therapy of human cytomegalovirus (HCMV) infection, since HCMV-encoded proteins are abundantly expressed on the surface of infected cells. Here we show that a BiTE antibody construct directed against HCMV glycoprotein B (gB) and CD3 efficiently triggers T cells to secrete IFN-γ and TNF upon co-culture with fibroblasts infected with HCMV strain AD169, Towne or Toledo. Titration of gB expression levels in non-infected cells confirmed that already low levels of gB are sufficient for efficient triggering of T cells in presence of the BiTE antibody construct. Comparison of redirecting T cells with the bispecific antibody versus a chimeric antigen receptor (CAR) based on the same scFv showed a similar sensitivity for gB expression. Although lysis of infected target cells was absent, the BiTE antibody construct inhibited HCMV replication by triggering cytokine production. Notably, even strongly diluted supernatants of the activated T cells efficiently blocked the replication of HCMV in infected primary fibroblasts. In summary, our data prove the functionality of the first BiTE antibody construct targeting an HCMV-encoded glycoprotein for inhibiting HCMV replication in infected cells.

Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110.

BACKGROUND: Bispecific T cell engager (BiTE®) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-ß), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells. METHODS: The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells. FINDINGS: An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-ß and PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not. CONCLUSIONS: Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct.

Side-by-side analysis of five clinically tested anti-EpCAM monoclonal antibodies.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is frequently and highly expressed on human carcinomas. The emerging role of EpCAM as a signalling receptor and activator of the wnt pathway, and its expression on tumor-initiating cells, further add to its attractiveness as target for immunotherapy of cancer. Thus far, five conventional monoclonal IgG antibodies have been tested in cancer patients. These are murine IgG2a edrecolomab and its murine/human chimeric IgG1 antibody version, and humanized, human-engineered and fully human IgG1 antibodies 3622W94, ING-1, and adecatumumab (MT201), respectively. Here we compared all anti-EpCAM antibodies in an attempt to explain differences in clinical activity and safety. METHODS: We recombinantly produced all antibodies but murine edrecolomab and investigated them for binding affinity, EpCAM epitope recognition, ADCC and CDC, and inhibition of breast cancer cell proliferation. RESULTS: ING-1 and 3622W94 bound to EpCAM with much higher affinity than adecatumumab and edrecolomab. Edrecolomab, ING-1, and 3622W94 all recognized epitopes in the exon 2-encoded N-terminal domain of EpCAM, while adecatumumab recognized a more membrane proximal epitope encoded by exon 5. All antibodies induced lysis of EpCAM-expressing cancer cell lines by both ADCC and CDC with potencies that correlated with their binding affinities. The chimeric version of edrecolomab with a human Fcγ1 domain was much more potent in ADCC than the murine IgG2a version. Only adecatumumab showed a significant inhibition of MCF-7 breast cancer cell proliferation in the absence of complement and immune cells. CONCLUSION: A moderate binding affinity and recognition of a distinct domain of EpCAM may best explain why adecatumumab showed a larger therapeutic window in cancer patients than the two high-affinity IgG1 antibodies ING-1 and 3622W94, both of which caused acute pancreatitis.

Highly efficient elimination of colorectal tumor-initiating cells by an EpCAM/CD3-bispecific antibody engaging human T cells.

With their resistance to genotoxic and anti-proliferative drugs and potential to grow tumors and metastases from very few cells, cancer stem or tumor-initiating cells (TICs) are a severe limitation for the treatment of cancer by conventional therapies. Here, we explored whether human T cells that are redirected via an EpCAM/CD3-bispecific antibody called MT110 can lyse colorectal TICs and prevent tumor growth from TICs. MT110 recognizes EpCAM, a cell adhesion molecule expressed on TICs from diverse human carcinoma, which was recently shown to promote tumor growth through engagement of elements of the wnt pathway. MT110 was highly potent in mediating complete redirected lysis of KRAS-, PI3 kinase- and BRAF-mutated colorectal TICs, as demonstrated in a soft agar assay. In immunodeficient mice, MT110 prevented growth of tumors from a 5,000-fold excess of a minimally tumorigenic TIC dose. T cells engaged by MT110 may provide a potent therapeutic means to eradicate TICs and bulk tumor cells derived thereof.

Heterogeneous nuclear ribonucleoprotein H blocks MST2-mediated apoptosis in cancer cells by regulating A-Raf transcription.

A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. Here, we show that A-Raf prevents cancer cell apoptosis contingent on the expression of the heterogeneous nuclear ribonucleoprotein H (hnRNP H) splice factor, which is required for the correct transcription and expression of a-raf. Apoptosis was prevented by A-Raf through sequestration and inactivation of the proapoptotic MST2 kinase. Small interfering RNA-mediated knockdown of hnRNP H or A-Raf resulted in MST2-dependent apoptosis. In contrast, enforced expression of either hnRNP H or A-Raf partially counteracted apoptosis induced by etoposide. In vivo expression studies of colon specimens corroborated the overexpression of hnRNP H in malignant tissues and its correlation with A-Raf levels. Our findings define a novel mechanism that is usurped in tumor cells to escape naturally imposed apoptotic signals.

Antitumor activity of an EpCAM/CD3-bispecific BiTE antibody during long-term treatment of mice in the absence of T-cell anergy and sustained cytokine release.

muS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. MT110, its human-specific analog, is in a clinical phase 1 trial for treatment of patients with adenocarcinoma of the lung or gastrointestinal tract. Recent studies have shown a therapeutic window for muS110, have explored single-dose toxicity of muS110, and have found that a 1-week low-dose treatment dramatically increased the tolerability of mice to very high doses of muS110 (Cancer Immunol. Immunother. 2009;58:95-109). Here we analyzed the impact of long-term, high-dose treatment of mice with muS110 on antitumor activity and functionality of T cells. After an initial self-limiting cytokine release, the 1-week adaptation period effectively blunted further cytokine production in response to a subsequent high-dose treatment with muS110. The much-increased tolerability of mice adapted to muS110 was not because of anergy of T cells. T cells isolated from chronically muS110-treated mice fully retained their cytotoxic potential, proliferative capacity, and responsiveness to stimulation by either muS110 or anti-CD3/anti-CD28/interleukin-2 when compared with T cells from control mice. Unimpaired T-cell performance was also evident from the effective prevention of orthotopic 4T1 breast tumor outgrowth in mice treated long term with escalating doses of muS110. Finally, we show that muS110 and MT110 recognize orthologous epitopes on mouse and human EpCAM proteins, suggesting that the target-related safety profile of muS110 in mice may be predictive for MT110 in humans.

The emerging role of EpCAM in cancer and stem cell signaling.

Initially discovered as a dominant antigen on colon carcinomas, the epithelial cell adhesion molecule (EpCAM) was considered a mere cell adhesion molecule and reliable surface-binding site for therapeutic antibodies. Recent findings can better explain the relevance of EpCAM's high-level expression on human cancers and cancer propagating cells, and its negative prognostic potential for survival of patients with certain cancers. EpCAM has oncogenic potential and is activated by release of its intracellular domain, which can signal into the cell nucleus by engagement of elements of the wnt pathway.

Nuclear signalling by tumour-associated antigen EpCAM.

EpCAM was found to be overexpressed on epithelial progenitors, carcinomas and cancer-initiating cells. The role of EpCAM in proliferation, and its association with cancer is poorly explained by proposed cell adhesion functions. Here we show that regulated intramembrane proteolysis activates EpCAM as a mitogenic signal transducer in vitro and in vivo. This involves shedding of its ectodomain EpEX and nuclear translocation of its intracellular domain EpICD. Cleavage of EpCAM is sequentially catalysed by TACE and presenilin-2. Pharmacological inhibition or genetic silencing of either protease impairs growth-promoting signalling by EpCAM, which is compensated for by EpICD. Released EpICD associates with FHL2, beta-catenin and Lef-1 to form a nuclear complex that contacts DNA at Lef-1 consensus sites, induces gene transcription and is oncogenic in immunodeficient mice. In patients, EpICD was found in nuclei of colon carcinoma but not of normal tissue. Nuclear signalling of EpCAM explains how EpCAM functions in cell proliferation.

Glycosylation is crucial for stability of tumour and cancer stem cell antigen EpCAM.

Epithelial cell adhesion molecule EpCAM is strongly over-expressed in a variety of carcinomas where it is involved in signalling events resulting in increased expression of target genes such as c-Myc, cyclins and others, eventually conferring cells an oncogenic phenotype. However, EpCAM is also expressed in a series of healthy epithelia, albeit generally to a far lesser extend. We have uncovered differential glycosylation of EpCAM as a means to discriminate normal from malignant tissues. EpCAM was hyperglycosylated in carcinoma tissue as compared with autologous normal epithelia. All three N-glycosylation consensus sequences within EpCAM's extracellular domain were used in human and murine cells. We show that glycosylation at asparagine198 is crucial for protein stability. Mutants of EpCAM that substitute asparagine198 for alanine showed a decreased overall expression and half-life of the molecule at the plasma membrane. This is of considerable importance with respect to EpCAM variants expressed in normal tissue, where it might reveal to be less stable and thus may have repercussions on functionality.

Carcinoma-associated eIF3i overexpression facilitates mTOR-dependent growth transformation.

Molecular processes controlling mRNA translation are complex, multilayered, and their deregulation can lead to cancer pathogenesis. Eukaryotic initiation factor 3 (eIF3) is involved in the initiation process of protein translation and overexpression of its subunit eukaryotic translation initiation factor i (eIF3i) has been observed in carcinomas. Nevertheless, the potential role of eIF3i in carcinogenesis is poorly understood. Here, we show that in vitro overexpression of human eIF3i resulted in cell size increase, proliferation enhancement, cell-cycle progression, and anchorage-independent growth. Without external stimuli, eIF3i overexpressing cells arrested in G1/G0 phase, demonstrating the requirement of additional growth signals. Inhibition of the kinase mTOR, a key player in the integration of nutrition and growth signals into protein synthesis, with rapamycin reduced serine phosphorylation of eIF3i and resulted in a loss of anchorage-independent growth. Thus, eIF3i overexpression fosters the integration of growth signals by mTOR into the mRNA translation process, promoting protein synthesis and tumor growth.

The tumour-associated antigen EpCAM upregulates the fatty acid binding protein E-FABP.

The epithelial cell adhesion molecule, EpCAM, is a transmembrane glycoprotein associated with both benign and malignant proliferation. In cancer cells, expression levels of this tumour-associated antigen correlate positively with the grade of dysplasia and are also a negative prognostic factor for breast cancer patients. De novo expression of EpCAM resulted in the rapid upregulation of the proto-oncogene c-Myc along with enhanced cell proliferation and metabolism. Here, we analyzed the effects of EpCAM onto the proteome of epithelial cells. The epidermal fatty acid binding protein, E-FABP, was identified as a new EpCAM-regulated protein. E-FABP is a major target of c-Myc and was rapidly upregulated upon induction of EpCAM. Additionally, E-FABP levels correlated with the amount of EpCAM in permanent squamous cell carcinoma lines and in vivo in primary head and neck carcinomas. Taken together, these results provide further evidence for the direct involvement of EpCAM in signalling processes, gene regulation, and cellular metabolism supporting its important role in tumour biology.

The carcinoma-associated antigen EpCAM upregulates c-myc and induces cell proliferation.

Epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein expressed on adenomatous and simple epithelia, where it is involved in homophilic adhesion at the basolateral membrane. Carcinomas strongly overexpress EpCAM through an, as yet, unknown mechanism. Interestingly, otherwise EpCAM-negative squamous epithelia are seen to express EpCAM concomitant with their transformation and de-differentiation. The amount of EpCAM and the number of expressing cells both increase with the grade of dysplasia. Despite an important amount of data correlating the expression of EpCAM with cellular proliferation and de-differentiation, such as the coexpression with Ki-67, a marker for proliferation, it is unknown whether EpCAM may directly contribute to carcinogenesis. Here, we show that EpCAM has a direct impact on cell cycle and proliferation, and the ability to rapidly upregulate the proto-oncogene c-myc and cyclin A/E. Human epithelial 293 cells as well as murine NIH3T3 fibroblasts expressing EpCAM had a decreased requirement for growth factors, enhanced metabolic activity and colony formation capacity. Importantly, the inhibition of EpCAM expression with antisense mRNA led to a strong decrease in proliferation and metabolism in human carcinoma cells. Moreover, domain swapping experiments demonstrated that the intracellular part of EpCAM is necessary and sufficient to transduce the effects described. Thus, the data presented here highlight the role of EpCAM, demonstrating for the first time a direct link to cell cycle and proliferation.

Targeted gene expression using a 1.1 kilobase promoter fragment of the tumour-associated antigen EpCAM.

The epithelial cell adhesion molecule EpCAM is over- or de novo expressed during carcinogenesis. EpCAM expression correlates with increased proliferation and dedifferentiation. Recently, we reported the cloning of a 1.1 kilobase fragment upstream of the epcam gene and demonstrated its specific transcriptional activity. Here, we analyzed the potential of this fragment for targeted gene expression. The fragment was used to regulate the expression of the green fluorescent protein (GFP) and HSV-1 thymidine kinase (HSV-TK), as a model therapeutic gene. Transfection of the pEpProm-control or pEpProm-GFP plasmids resulted in the expression of functional GFP and HSV-TK proteins specifically in EpCAM-positive cells. Expression levels of both proteins correlated with the amount of EpCAM. Additionally, the targeted expression of HSV1-TK transferred a marked sensitivity to ganciclovir treatment in EpCAM-positive HEK293-EBNA1 and SkBr3 carcinoma cells. The EpCAM promoter fragment is, thus, a novel tool to allow for the transcription of therapeutic genes, specifically, in EpCAM-positive carcinomas.

The Carcinoma-associated Antigen EpCAM Induces Glyoxalase 1 Resulting in Enhanced Methylglyoxal Turnover.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is a homophilic adhesion molecule expressed de novo on a variety of epithelial tumors. Overexpression of EpCAM results in enhanced proliferation and rapid induction of the proto-oncogene c-myc. MATERIALS AND METHODS: The novel proteomics-based fluorescence difference gel electrophoresis (DIGE technology) was used to study EpCAM effects on the proteome of human epithelial cells. RESULTS: DIGE analysis resulted in the identification of five proteins with a significantly changed regulation ranging from -1.3 to +5.8-fold. One of the identified proteins, namely glyoxalase 1, experienced a shift in the isoelectric point from pH 5.2 to 5.0 upon EpCAM expression. This shift correlated with a gain of enzymatic activity of glyoxalase 1 resulting in an enhanced methylglyoxal turnover. CONCLUSION: We show the potential of the DIGE technology to rapidly and quantitatively analyze proteomes for changed expression levels and, importantly, posttranslational modifications. Furthermore, we describe new targets of the carcinoma antigen EpCAM including glyoxalase1.

Cloning and characterisation of a 1.1 kb fragment of the carcinoma-associated epithelial cell adhesion molecule promoter.

The epithelial cell adhesion molecule (EpCAM) is a transmembrane protein associated with a variety of carcinomas, where EpCAM is often strongly up-regulated or, as in the case of squamous cell carcinomas, de novo expressed. The molecular mechanisms underlying the transcriptional regulation of EpCAM are poorly understood. So far, a 570 bp fragment has been cloned and shown to have specific transcriptional activity, which was negatively-regulated upon the induction of the transcription factor NF-kappa B. In the present study we have cloned a 1100 bp fragment of the EpCAM promoter containing the 570 bp fragment and additional 550 bp upstream. We demonstrate that both fragments have strong synergistic effects with respect to transcriptional activity in EpCAM-positive cells. Furthermore, the 1100 bp fragment was likewise negatively-regulated upon TNF alpha and IFN alpha treatment, thus retaining silencer sequences.

Tumor-specific glycosylation of the carcinoma-associated epithelial cell adhesion molecule EpCAM in head and neck carcinomas.

The tissue-specific glycosylation of the carcinoma (CA)-associated antigen epithelial cell adhesion molecule (EpCAM) was studied in 60 patients suffering from head and neck CAs, and 26 pairs of autologous healthy thyroid and CA biopsies. EpCAM was glycosylated in all tumor samples in which its expression was detectable (73%). Additionally, in 80.7% of patients, tumor-derived EpCAM was heavily glycosylated while EpCAM derived from autologous thyroid was not (76.2%) or weakly (23.8%). Four cases showed a similar glycosylation pattern (15.3%) and one case displayed a reverse pattern (3.8%). Additionally, the expression and glycosylation of EpCAM were assessed in tumor adjacent and distant tissue. EpCAM was glycosylated in tumor-adjacent while it was not or only weakly expressed in tumor distant tissue where it was unglycosylated. Thus, EpCAM is differentially glycosylated in healthy tissue and tumor cells of the head and neck area.