Objective
We evaluated the expression of epithelial cell adhesion molecule (EpCAM) and the potential of MT201 (adecatumumab), a human-monoclonal-antibody that targets EpCAM against chemotherapy-resistant ovarian disease.
Study Design
EpCAM expression was evaluated by real-time polymerase chain reaction and flow cytometry. Sensitivity to MT201 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity was tested in 4-hour chromium-release assays. The effect of interleukin-2 on MT201 ADCC was also studied.
Results
High messenger RNA expression by real-time polymerase chain reaction and high EpCAM surface expression by flow cytometry was detected in 71% of ovarian cancers (5 of 7 cell lines). Although these cell lines were highly resistant to complement-dependent cytotoxicity and natural killer-dependent cytotoxicity in vitro (range of killing, 0–7%), EpCAM-positive cell lines showed high sensitivity to MT201 ADCC (range of killing, 27–66%). Incubation with interleukin-2 further increased the cytotoxic activity against EpCAM-positive ovarian cancer cell lines.
Conclusion
MT201 may represent a novel, potentially highly effective treatment option for patients with ovarian carcinoma whose body is harboring disease refractory to chemotherapy.
Ovarian cancer is the gynecologic malignancy with the highest mortality rate; 21,550 new cases and 14,600 deaths had been estimated for 2009. Most ovarian cancers are diagnosed in advanced stages. Despite aggressive surgical treatment and chemotherapy, the 5-year survival rate of patients with advanced stage disease is 30%. Although the disease responds to the initial chemotherapy, it ultimately becomes resistant to the treatment. Thus, there is great interest in the development of targeted agents active against chemotherapy-resistant ovarian disease.
Epithelial cell adhesion molecule (EpCAM; also referred to as EGP-40, Trop-1, 17-1A, KSA, KS1/4, AUA1, GA733-2, and CD326) has been shown to be overexpressed in ovarian cancer. EpCAM is a 40-kd surface glycoprotein that was first discovered in 1979. The EpCAM gene is located on chromosome 4q and contains 9 exons and consists of an extracellular domain with 2 EGF-like repeats, a transmembrane, and a short cytoplasmic domain. EpCAM is expressed at low levels on the basolateral and intercellular surface of simple, pseudostratified, and transitional epithelia, which includes most epithelial tissues in the female genital tract. The homogenous distribution of EpCAM on the tumor cell, its glycosylation, and the level of expression help differentiate tumor from normal cells. Indeed, most neoplastic epithelial cells overexpress EpCAM, as do 85% of adenocarcinomas and 72% of squamous cell carcinomas. EpCAM is believed to contribute to signaling, cell migration, proliferation, and differentiation. It promotes cell adhesion through a calcium-independent mechanism ; the formation of EpCAM-mediated adhesions have a negative regulatory effect on adhesions that are mediated by cadherins. Consistent with this view, EpCAM silencing with small interfering RNA may led to a reduction in proliferation, migration, and invasion.
Importantly, because of its localization on the cell surface of carcinomas, EpCAM is an attractive target for immunotherapy. Edrecolomab (Panorex), a chimeric murine anti-EpCAM immunoglobulin G2a (IgG2a) antibody, was shown to improve overall and disease-free survival in patients with Dukes C colon cancer and minimal residual disease. It subsequently gained approval in Germany as an adjuvant monotherapy in the treatment of colon carcinoma and was taken off the market only after the introduction of 5-fluorouracil with leucovorin in colon carcinoma led to even better survival results.
Adecatumumab (MT201) is a fully human, recombinant anti-EpCAM monoclonal antibody (mAb), which acts mainly through antibody-dependent cellular cytotoxicity (ADCC). Compared with the murine antibody edrecolomab, MT201 shows a longer half-life and reduced immunogenicity. Unlike the previous murine high-affinity anti-EpCAM antibodies, MT201 is a low-to-intermediate affinity antibody. The high-affinity antibodies were associated with significant toxicities in phase I clinical trials. MT201, however, appears to be well tolerated, has been tested in phase II trials in metastatic breast and early-stage prostate cancer, and currently is being evaluated in a phase IB trial in combination with docetaxel and another phase II study in patients who are undergoing liver resection for colorectal cancer metastases.
In this investigation, we evaluated the potential value of EpCAM as a novel target against ovarian cancer by studying its expression at both messenger RNA (mRNA) and protein level in multiple, biologically aggressive ovarian cancer cell lines that have been established from patients whose body harbors chemotherapy-resistant disease.
Materials and Methods
Establishment of ovarian cancer cell lines
Study approval was obtained from the institutional review board, and all patients signed an informed consent form according to institutional guidelines. A total of 7 ovarian cancer cell lines were established after the sterile processing of samples from surgical biopsy specimens, as previously described. Patient characteristics regarding the tumor cell line of origin are described in Table 1 . All patients received a combination of carboplatin and paclitaxel as the primary chemotherapy regimen. Six of the 7 patients whose cells were used for the establishment of cell lines demonstrated disease progression on chemotherapy. All 7 primary ovarian cancer cell lines were found highly resistant in vitro to multiple chemotherapy drugs, which included carboplatin, cisplatin, paclitaxel, doxorubicin, ifofosfamide, gemcitabine, and topotecan by Extreme Drug Resistant assays (Oncotech, Irvine, CA).
| Cell line | Histology | Age, y | Race | Stage | Grade |
|---|---|---|---|---|---|
| OSPC-ARK-1 | OSPC | 68 | C | IV | G3 |
| OSPC-ARK-2 | OSPC | 64 | C | IIIC | G3 |
| OSPC-ARK-3 | OSPC | 53 | C | IIIA | G3 |
| OSPC-ARK-4 | OSPC | 33 | C | IV | G3 |
| OSPC-ARK-5 | OSPC | 55 | C | IV | G3 |
| CC-ARK-1 | CC | 42 | C | IIIC | G3 |
| CC-ARK-2 a | CC | 32 | C | IC | G3 |
a With the exception of CC-ARK-2, all tumor biopsy specimens from which ovarian cancer cell lines were established were collected at the time of tumor recurrence after multiple chemotherapy regimens; primary ovarian cancer cell lines were not selected against chemotherapy drugs in vitro.
Quantitative real-time polymerase chain reaction (q-rtPCR)
RNA isolation from a total of 7 primary ovarian carcinoma cell lines was performed with TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Quantitative PCR was performed with a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA), according to the manufacturer’s recommended protocol to evaluate the expression of EpCAM in all samples. The primers and probe for EpCAM were obtained from Applied Biosystems. The comparative threshold cycle ( C T ) method (Applied Biosystems) was used to determine gene expression in each sample relative to the value that was observed in the lowest nonmalignant ovarian epithelial cell sample, with glyceraldehyde-3-phosphate dehydrogenase (Assay ID Hs99999905_m1) RNA as internal control.
Flow cytometry
The humanized anti-EpCAM mAb MT201 (Micromet, Munich, Germany) was used for flow cytometry studies. The chimeric anti-CD20 mAb rituximab (Rituxan; Genentech, San Francisco, CA) was used as antibody isotype control for MT201. A goat anti-human F(ab’) 2 immunoglobulin (BioSource International, Camarillo, CA) was used as a secondary reagent. Analysis was conducted with a FACScalibur flow cytometer with Cell Quest software (Becton Dickinson, Franklin Lakes, NJ).
ADCC measurements
A standard 4-hour chromium release assay was performed to measure the cytotoxic reactivity of Ficoll-Hypaque separated peripheral blood lymphocytes (PBL) that were obtained from several healthy donors against all 7 ovarian cancer cell lines. In most of the experiments, the release of chromium from the target cells was measured as evidence of tumor cell lysis after exposure of tumor cells to a concentration 5 μg/mL of MT201. Controls included the incubation of target cells alone or with PBL or mAb separately. The chimeric IgG1 anti-CD20 monoclonal antibody rituximab (Rituxan; Genentech) was used as antibody isotype control for MT201 in all bioassays.
Interleukin-2 (IL-2) enhancement of ADCC
To investigate the effect of IL-2 on MT201-mediated ADCC, effector PBL were incubated for 4 hours at 37°C at a final concentration of IL-2 (Aldesleukin; Chiron Therapeutics, Emeryville, CA), which ranged from 50–100 IU/mL in 96-well microtiter plates. Target cells were primary ovarian cell lines that had been exposed to MT201 (concentration, 5 μg/mL), whereas controls included the incubation of target cells alone or with PBL in the presence or absence of IL-2 or mAb, respectively. Rituximab was used as an isotype control mAb.
Test for complement-mediated target cell lysis and for inhibition by γ-immunoglobulin
A standard 4-hour chromium-release assay identical to those used for ADCC assays was used, except that human serum in a dilution of 1:2 was added in place of the effector cells. This human serum was used as a source of complement to test for complement-mediated target cell lysis. To test for the possible inhibition of ADCC against ovarian cancer cell lines by physiologic human serum concentrations of γ-immunoglobulin, heat-inactivated (56°C for 60 minutes) human serum was diluted 1:2 before being added in the presence or absence of effector PBL. In some experiments, non–heat-inactivated human serum (diluted 1:2) was added in the presence of effector PBL. Controls included the incubation of target cells alone or with either lymphocytes or mAb separately. Rituximab was used as isotype control mAb.
Statistical analysis
Q-rtPCR data were evaluated with unequal-variance t test for ovarian carcinoma vs normal ovarian epithelial cells difference. Differences in EpCAM expression by flow cytometry were analyzed by the unpaired t test; a probability value of < .05 among samples was considered to be significant. Kruskal-Wallis test and χ 2 analysis were used to evaluate differences in MT201-induced ADCC levels in primary tumor cell lines. Statistical analysis was performed with SPSS statistical software (version 15; SPSS, Inc, Chicago, IL).
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