In his introductory remarks, G Riethmueller (Munich, Germany) pointed out that the first human tumour-associated antigen identified with monoclonal antibodies (mAb) was in fact EpCAM or 17-1A antigen (Herlyn et al, 1979). Moreover, the first monoclonal antibody ever applied for human cancer therapy was murine mAb 17-1A recognising EpCAM (Sears et al, 1982, 1984). In 1994, mAb17-1A (later named edrecolomab and Panorex®) was also the first to show clinical efficacy in a human cancer indication in terms of prolonged overall survival (Riethmüller et al, 1994). It was then almost 30 years after the first generation of an EpCAM-specific monoclonal antibody that an international community gathered in Bavaria to discuss for the first time the most recent advances in the field of EpCAM research and clinical utility.

EpCAM has meanwhile advanced to an established epithelial cell marker used in pathological examinations. Likewise, EpCAM can now be considered to be one of the most frequently and most intensely expressed tumour-associated antigens known. It is found expressed on a great variety of human adenocarcinoma and squamous cell carcinoma (Went et al, 2004). Very recent immunohistochemistry (IHC) studies analysed fairly large sample numbers from patients with breast, prostate, ovarian, lung, colon, renal, and gastric cancer (Spizzo et al, 2004, 2006b; Went et al, 2005, 2006). These data impressively underscore the potential utility of EpCAM as immunotherapeutic target for treatment of the most frequent human cancers.

At least seven different immunotherapeutic approaches are currently in clinical trials that target EpCAM (see Table 1). Some of them have already progressed to the level of phase II and pivotal trials, indicating that initial safety questions with EpCAM-directed therapeutic approaches have been successfully addressed in preceding phase 1 studies and that the assessment of clinical efficacy is now of primary interest.

Only recently, research on EpCAM started to focus on its role in carcinogenesis. In 2004, first evidence for a direct role of EpCAM in growth-promoting nuclear signalling was reported (Munz et al, 2004). These data and results published by the group of Gillanders (Osta et al, 2004) revived research on the biological function of EpCAM, which is now delivering breakthrough results.

The present meeting review is focused on new data investigating the significance of EpCAM for cancer biology and therapy, and can therefore not adequately cover all presentations given at the symposium. We apologise to those attendees whose presentations are not mentioned or not fully covered. The majority of the meeting presentations can be viewed at www.epcam-symposium.de.

A highlight of the symposium was new data presented by M Munz (Munich, Germany, manuscript submitted) that unravelled the entire pathway of EpCAM signalling from the cell membrane into the nucleus. Epithelial-specific cell adhesion activation molecule signalling was found induced either by soluble EpCAM or through zones of cell–cell contact, suggesting that oligomerisation is a trigger for EpCAM signalling. These signals led to cleavage of EpCAM by proteases, releasing an intracellular peptide termed EpIC. Involvement of proteases in EpCAM signalling was supported by both their co-precipitation with EpCAM and by the inhibitory effects of specific small molecule protease inhibitors, which also reduced EpCAM-mediated gene expression events.

Two-hybrid screening identified adaptor proteins as intracellular receptors for EpIC allowing the formation of a multiprotein signalling complex shown to be involved in target gene induction and growth-promoting effects. Multicolour immunofluorescence analysis impressively demonstrated cytoplasmic/nuclear redistribution of all the involved proteins depending on EpCAM signalling. An antibody to EpIC was found to give a speckled staining inside the nucleus of stimulated cells, indicating an association of the EpCAM signalosome with specific chromatin sites. With specific inhibitors of EpCAM cleavage, a pharmacological means is given to interfere with EpCAM signalling in tumour cells. Future studies are needed to understand how EpCAM signalling is regulated in normal vs malignant cells.

C Klein (Munich, Germany), a pioneer in the study of single DTC for genomic aberrations and transcriptional patterns (Klein et al, 2002a, 2002b), investigated subpopulations of tumour cells as found in bone marrow biopsies of prostate cancer patients. In these patients staged as M0 (no metastasis), BR (biochemical PSA relapse), and M1 (established metastasis), EpCAM-positivity of bona fide tumour cells significantly increased with progression from M0 to BR, and further to M1 stages from 9 to 16–33%. Epithelial-specific cell adhesion activation molecule⁺ tumour cells had double the amount of chromosomal aberrations than CK⁺ tumour cells, and these aberrations affected different chromosome locations. There was only a small overlap between EpCAM⁺ and CK⁺ DTC populations of 9.5%. Epithelial-specific cell adhesion activation molecule thus defined a subpopulation of DTC in prostate cancer patients that – unlike CK⁺ DTC – already expanded during biochemical relapse and had a phenotype different from that of CK⁺ tumour cells.

G Gastl (Innsbruck, Austria) reviewed the expression of EpCAM in human breast cancer and its strong negative correlation with survival parameters in node-positive patients (Spizzo et al, 2004). Efforts for standardising the immunohistochemical staining and evaluation procedure were explained highlighting an advanced scoring system that reflected both frequency, and intensity of staining. A similar negative correlation between EpCAM expression and survival as previously observed by the group for breast and gall bladder cancer (Varga et al, 2004) was also now observed with ampullary carcinoma of the pancreas (Fong et al, 2006) and for squamous cell cancer of head and neck.

N Stoecklein (Duesseldorf, Germany) reported a negative correlation between EpCAM expression and overall survival in patients with squamous cell carcinoma of the oesophagus, a very deadly disease with no established systemic treatment option. Although normal epithelium showed no EpCAM expression, up to 41% of patients showed high-level expression with a strong negative prognostic impact (P=0.0002) (Stoecklein et al, 2006).

A number of studies aim at the understanding of the regulation of EpCAM at the transcriptional level. These studies include deletion analysis of the EpCAM promoter and identification of transcription factors governing EpCAM expression (O Gires, Munich, W Gommans, and I van der Gun, The Netherlands) and analysis of the methylation and amplification status of the EpCAM gene (G Spizzo, Innsbruck, Austria). No differences in promoter methylation could be found in breast cancer vs normal tissue samples, albeit studies in breast cancer cell lines suggested a role for methylation in the regulation of EpCAM expression (Spizzo et al, 2006a).

P Baeuerle (Carlsbad, USA) described adecatumumab (MT201), a fully human anti-EpCAM antibody (Naundorf et al, 2002) that currently is in three ongoing clinical trials: two phase II studies with metastatic breast cancer and early-stage prostate cancer patients, respectively, and a phase I study testing the safety of a combination with taxotere. A completed phase I study in hormone-resistant prostate cancer patients has shown that the human antibody was well tolerated at all doses tested, has not reached a maximum tolerated dose at 6mgkg⁻¹, and showed no signs of pancreatitis, which had been an issue with two high-affinity anti-EpCAM mAbs. Preclinical examination has shown that adecatumumab mainly acts through antibody-dependent cellular (ADCC) and complement-dependent cytotoxicity (CDC), and shows high antitumour activity in a lung metastasis mouse model when equipped with a murine Fc portion of the γ2A subtype, which is better compatible with the murine immune system than the human γ1 subtype. The speaker also presented preclinical data on MT110, a single-chain EpCAM/CD3-bispecific antibody construct of the BiTE class (Brischwein et al, 2006). In immunodeficient mice, very low doses of MT110 led to the eradication of established tumours derived from human cancer cell lines mixed with human T cells, as well as ovarian cancer tissue of patients. In the latter case, the sole source of human T cells was the xenograft itself, indicating that MT110 can penetrate human tumour xenografts after intravenous (i.v.) injection and reactivate the low numbers of tumour-resident T cells to effectively eliminate the metastatic tissue.

H Lindhofer (Munich, Germany) reviewed preclinical and clinical data of catumaxomab, a hybrid mouse IgG2a/rat IgG2b antibody that is trispecific for EpCAM, CD3 and -via its Fc portion- for antigen-presenting as well as a variety of other cytotoxic immune cells. In mouse tumour models the trioma format shows high antitumour activity and survival of which part is due to a vaccination effect (Lindhofer et al, 1996). When intraperitoneally (i.p.) given to ovarian cancer patients suffering from ascites and peritoneal carcinomatosis, a pronounced clinical activity was observed that in a phase I trial 21 out of 23 patients were freed of malignant ascites. A rapid clearance of essentially all EpCAM-positive tumour cells in ascites fluid and an expansion of T-cell numbers were observed, which was followed by a retraction of immune cell counts.

A pivotal study of catumaxomab in ovarian cancer patients with malignant ascites has already enroled more than 250 patients. A phase II study is ongoing with gastric cancer patients who are treated during gastric surgery for prevention of peritoneal carcinomatosis. Hence, catumaxomab appears as a very promising biological agent for the local treatment and prevention of peritoneal carcinomatosis.

A variation of the trispecific approach was presented by G Moldenhauer (Heidelberg, Germany). Hybrid antibodies containing one half of the EpCAM-specific antibody HEA125 and one half of the anti-CD3 antibody OKT3 were generated by the hybrid-hybridoma technique. Preclinical experiments showed that T cells isolated from malignant ascites support a redirected lysis of tumour cells in vitro by the HEA125 × CD3 trispecific antibody. Ten ovarian cancer patients were treated in a small clinical study with a 1mg dose of antibody. Inhibition of ascites production was observed in eight out of 10 patients. A dramatic several thousand-fold increase in TNF-α was measured in ascites, indicating a very strong local immune stimulation. For selective recruitment of activated neutrophils and macrophages, a second construct was generated using mAb HEA125 that combines the anti-EpCAM mAb with the anti-CD64/FcγRI mAb 197. Treatment of one ovarian cancer patient with 6 × 1mg HEA125 × 197 also reduced ascites and CA−125. Three clinical trials are in planning stage exploring dose escalations of HEA125 × CD3 and HEA125 × 197 and a combination thereof in ovarian cancer patients with malignant ascites.

U Zangemeister-Wittke (Bern, Switzerland) reported on the development of an EpCAM-specific immunotoxin called Proxinium (4D5MOC31-ETA) that currently is in a pivotal phase II/III trial for local treatment of head & neck cancer. An earlier phase I/II study has shown a tumour control rate of 88% in which 25% of injected lesions showed a complete response. Median survival of treated patients was 301 days compared with 125 days for a historic control group. The same antibody construct is now being developed for local treatment of bladder cancer (here called Vicinium®). The immunotoxin uses a stability-engineered single-chain humanised anti-EpCAM antibody fused to a subunit of the bacterial Pseudomonas exotoxin. The linker contains a furin cleavage site that allows for release within the endosome of the toxin after EpCAM binding and endocytosis. A conformational change of the cleaved exotoxin enables its cytosolic entry and highly efficient inhibition of the cell's protein synthesis. A novel EpCAM-directed immunotoxin is under development that has the furin cleavage site replaced by a site cleaved through matrix metalloproteinases-2 and -9, as are selectively expressed by tumour cells. This enables a dual targeting that may increase the immunotoxin's therapeutic window. In vitro data support that cell lines lacking MMP−9 and −2 are much less vulnerable to the immunotoxin, and that specific MMP inhibitors partially protect cells expressing the proteases from the immunotoxin. Another EpCAM-directed therapy presented by the speaker uses liposomes with single-chain anti-EpCAM antibodies linked via polyethylene glycol moieties. These long-lived liposomes are being loaded with a mix of anti-apoptotic antisense molecules specific for Bcl-2 and Bcl-XL or with doxorubicin. Xenotransplant mouse models designed for studying the targeting of [³H]-labelled liposomes and antitumour activity support the usefulness and potency of this approach and a combination of liposomes with anti-apoptotic and chemotherapeutic payloads.

D Herlyn (Philadelphia, USA) reviewed progress on using EpCAM as a vaccine to elicit tumour-specific T-cell and humoral immune responses. A variety of approaches were tested in small un-controlled clinical trials that use for vaccination an anti-idiotypic antibody (BR3E4), the extracellular domain of EpCAM or EpCAM encoded by an adenovirus. Evidence for specific T-cell responses and antigen spreading could be obtained.

C Potten (Manchester, UK) set the stage for this discussion by reviewing in a very comprehensive manner the epithelial architecture, cellular dynamics, and fates of epithelial and stem cells in small intestine. P McLaughlin (Groningen, the Netherlands) investigated the consequences of targeting monoclonal antibodies to normal and tumour tissue in a transgenic murine model expressing human EpCAM. Antibodies were administered i.p. and murine tissue staining for bound antibodies performed 24–48h later. Doses of 1, 10, 100, and 1000μg antibody were given, of which a 10μg dose would corresponded to 0.4mgkg⁻¹ in humans. This covers well the range at which acute pancreatitis was seen with mAb ING-1 in man (>1mgkg⁻¹). With increasing dose, the human EpCAM-specific IgG1 antibody USB54 stained an increasing number of epithelial tissues in transgenic mice. At the low 10μg dose, the staining of pancreas, kidney and small intestine by the antibody was most prominent. Apparently, animals showed no adverse events even at the 1-mg dose despite the apparent binding of a potentially cytotoxic human IgG1 antibody to epithelial cells of many organs. It is unclear whether the co-expressed murine EpCAM in the transgenic model could prevent cytotoxic effects of the antibody. Animal models of this kind will in the future be helpful in deciphering the basis for a differential effect of antibodies on normal vs tumour tissue.

The most important insights into the accessibility of EpCAM on normal epithelia vs tumour cells may come from recent studies identifying a number of other proteins found in close association with EpCAM within the cell membrane. F Le Naour (Villejuif, France) presented evidence on the interaction of EpCAM with tetraspanins, a family of 4-transmembrane domain proteins with diverse biological functions. One of them, CD9, was shown by crosslinking experiments to directly bind EpCAM whereas other proteins including ADAM10, integrins, G proteins, and a novel CD9 binding protein (CD9P-1) were indirectly associated. Intriguingly, loss of CD9 expression correlates with metastasis (Si and Hersey, 1993; Miyake et al, 1996; Zheng et al, 2005) and, as reported at the symposium, a strict co-localisation of CD9 and EpCAM, as seen for membranes of normal colonic epithelium, was no longer found for tumour cell membranes.

M Zoeller (Heidelberg, Germany) reported an association of EpCAM with CD44 splice variants v4–v7 and tetraspanin D6.1A. Partial cholesterol depletion destroyed these complexes, indicating their co-localisation within membrane microdomains. A novel interaction of EpCAM was found with the serine-phosphorylated version of claudin-7, a protein found in tight junctions and the basolateral side of epithelia. Crosslinking experiments implied that interaction with EpCAM was direct and does not require the EGF-like and TY domain for binding (see Figure 1). Interestingly, claudin-7 prevented EpCAM oligomerisation, which may provide a means to suppress and control EpCAM signalling in normal epithelial tissue. This is corroborated by a co-localisation of EpCAM and claudin-7 in normal tissue.