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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2003 Jun 18;129(6):341–348. doi: 10.1007/s00432-003-0438-6

Cytotoxic activity of novel human monoclonal antibody MT201 against primary ovarian tumor cells

Wei Xiang 1, Pauline Wimberger 2,✉, Torsten Dreier 3, Joachim Diebold 4, Doris Mayr 4, Patrick A Baeuerle 3, Rainer Kimmig 2
PMCID: PMC12162085  PMID: 12819960

Abstract

Purpose

The epithelial cell adhesion molecule (Ep-CAM) is a clinically validated target for antibody-based therapy of cancer. The aim of this work was to evaluate the specific cytotoxic activity of a novel fully human Ep-CAM-specific IgG1 antibody, called MT201, against primary ovarian tumor cells and an ovarian tumor cell line.

Methods

The anti-tumor efficacy of MT201 was examined both in coculture of the ovarian cancer cell line OvCAR-3 and peripheral blood mononuclear cells (PBMCs) from healthy donors, and in primary metastatic tumor specimens freshly dissected from 21 patients with ovarian cancer using only the tumor-resident autologous effector cells. The extent of tumor cell depletion was determined by flow cytometry using Ep-CAM/CA-125 double-labeling or Ep-CAM labeling, both combined with propidium iodide uptake as cell lysis marker.

Results

MT201 at sub-µg/ml concentrations effectively eliminated OvCar-3 cells in the presence of PBMC. In freshly dissected tumor specimen, endogenous autologous immune cells could lyse, in a MT201-dependent fashion, Ep-CAM-positive tumor cells in 17 out of 21 patients showing an ex vivo response rate of 81%. In certain samples, up to 80% lysis of Ep-CAM-positive tumor cells by MT201 were observed after 16–30 h of incubation.

Conclusions

These data indicate that MT201 can effectively redirect tumor-resident effector cells against Ep-CAM-positive ovarian cancer cells and may therefore offer an effective therapy for ovarian cancer.

Keywords: Human monoclonal antibody, Ovarian cancer, Ex vivo tumor cell lysis, Ep-CAM

Introduction

Ovarian cancer ranks as the fourth most common cause of female cancer death in the United States with more than 26,000 new cases being recorded every year (Parker et al. 1997). At present, no effective screening method for detection of ovarian cancer is available and prognosis is very poor (Young al. 1993). About 3/4 of patients with primary ovarian cancer have an advanced stage of FIGO III or IV because patients are asymptomatic for a very long time or just show nonspecific symptoms like abdominal discomfort, ascites, or dyspepsia. Most of the patients with primary ovarian cancer present with a peritoneal carcinomatosis with lymph node involvement. Initial treatment usually includes radical surgical debulking followed by administration of platinum and paclitaxel, the standard therapy defined by the Gynecologic Oncology Group study (GOG) (McGuire et al. 1996).

Although the incorporation of paclitaxel and carboplatin as first-line chemotherapy has been shown to improve overall survival in patients with advanced ovarian cancer, the prognosis is still very poor with a 5-year survival rate of less than 45% in advanced stages (Averette et al. 1995). The relapse rate remains very high, mainly because of the development of resistance. Therefore, the development of new, effective second-line and first-line treatments for primary ovarian cancer remains a high priority. The limitation of chemotherapy is mainly due to a narrow therapeutic index between cancer and normal cells calling for therapeutics with less toxicity and higher specificity. The use of cytotoxic monoclonal antibodies could be one approach to improve prognosis of patients with ovarian cancer.

Within the last few years, different monoclonal antibody (mAb)-based strategies have been successfully applied to different malignancies. The murine anti-Ep-CAM monoclonal IgG2a edrecolomab (Panorex; 17–1A) was among the first monoclonal antibodies administered to humans for treatment of cancer (Sears et al. 1982). Murine IgG2a is the functional equivalent of human IgG1, which exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Clinical data with edrecolomab are somewhat controversial because two studies (the Riethmüller trial and a phase III US trial) showed efficacy, whereas a European phase III trial showed no efficacy (Riethmüller et al. 1998; Riethmüller et al. 1994; Fields et al. 2002; Punt et al. 2002). This antibody is relevant for the present study to the extent that it is validating the Ep-CAM target. Of note, no serious side effects were seen with edrecolomab in several thousand patients, supporting that the target is indeed differentially accessible between tumor and normal tissue (McLaughlin et al. 2000). Abdullaet al. found that chimeric and humanized antibodies were significantly better at mediating tumor lysis than their murine equivalents with all -effector populations tested (Abdullah et al. 1999).

Ep-CAM (epithelial cell adhesion molecule) is a highly conserved surface glycoprotein which is overexpressed in many carcinomas of different origin (Balzar et al. 1999). It is also specifically expressed on normal epithelia but apparently is not accessible there to i.v. administered antibody due to its restricted accessibility in highly structured epithelium (McLaughlin L et al. 2000). Based on the promising results with an Ep-CAM antibody in gastrointestinal malignancies, we tested in this study the potential of an anti-Ep-CAM antibody for treatment of ovarian cancer using an ex vivo model system. A novel fully human Ep-CAM-specific antibody, called MT201, that was derived from the repertoire of human IgD-positive B cells (Raum et al. 2001), was employed. MT201 is expected to have a much lower immunogenicity than the murine edrecolomab and was shown to have a much higher antibody-dependent cellular cytotoxicity (ADCC) than edrecolomab (Naundorf et al. 2002). Here, we observed that MT201 effectively eliminated a human ovarian carcinoma cell line as well as malignant cells in samples prepared from tumor tissue of patients with ovarian cancer. Tumor cell lysis did not require the addition of extra effector cells but apparently was mediated by antibody-dependent cellular cytotoxicity (ADCC) through tumor-resident autologous immune cells of patients.

Materials and methods

Cell line and PBMC preparation

The ovarian cancer cell line OvCAR-3 (ATCC No. HTB 161) was used to establish the flow cytometric detection of ovarian cancer cells over the background of blood-borne cells and other cell types found in tumors. It also allowed us to initially establish conditions for tumor cell elimination by MT201.

OvCAR-3 cells were grown in RPMI-1640, supplement with 10% fetal bovine serum (FBS), 1% Zienam (w/v) and 200 U/ml Nystatin, respectively. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2/95% air as a confluent monolayer. Exponentially proliferating cells were harvested with 0.05% trypsin and 0.02% EDTA, resuspended with RPMI-1640 and 10% FBS. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood of voluntary donors by Ficoll density centrifugation.

Patients

Tumor specimens from a total of 39 patients were collected. The material from the first 18 patients was required to establish tissue handling and cell preparation techniques, cell culture conditions, cytotoxic assay development, flow cytometric analysis, and standardized conditions for comparative analysis of antibody efficacy. Tumor samples from 21 patients with ovarian cancer were ultimately analyzed for the effect of MT201 on tumor cell depletion ex vivo. Tumor specimens were freshly dissected during surgery of patients with ovarian cancer. Only small tumor samples were dissected from peritoneal metastasis for the ex vivo experiments. The rest of dissected tumor mass was used for histological examination. The histology of corresponding patient samples are shown in Table 1.

Table 1.

Reactivity of MT201 with tumor cells in ovarian cancer tissue samples. The histological grading according to FIGO of patients and their response to MT201 as analyzed by flow cytometry is shown. -: <10% lysis of Ep-CAM+ cells; +: 10–20% lysis of Ep-CAM+ cells ; ++: 20–40% lysis of Ep-CAM+ cells; +++: 40–60% lysis of Ep-CAM+ cells; ++++: >60% lysis of Ep-CAM+ cells. *: no experiments were performed or no results could be obtained because of poor culture conditions

Patient number Histology Primary (p) or Recurrent (r) ovarian cancer CD45+ : Ep-CAM+ cell ratio Response to MT201 (16 h/30 h incubation)
16 Serous papillary adenocarcinoma, FIGO IIIc, G3 p 1:1 + / +
17 Extraovarian solid papillary ovarian cancer with psammom bodies, FIGO IV, G3 p 3:1 +++ / ++++
18 Papillary adenocarcinoma, FIGO IIIc, G3, 2× carboplatinum/paclitaxel r 2:1 ++ / *
19 Serous endometrioid, FIGO Ia, G2 p 4:1 +++ / *
20 Serous papillary adenocarcinoma, FIGO IIIc, G3 p 1:1 +++ / *
21 Serous papillary adenocarcinoma, FIGO IV, pT2a, G3 p 1:2 + / *
22 Serous papillary adenocarcinoma, FIGO IIIc, G3 p 12:1 ++++ / *
23 Solid, papillary carcinoma with psammom bodies, FIGO IIIc, G3 p 5:1 - / ++
24 Serous papillary adenocarcinoma, FIGO Ic, G3 p 2:1 - / *
25 Serous papillary adenocarcinoma, FIGO IIIc, G2, and renal cell carcinoma p 1:2 + / ++
27 Undifferentiated solid carcinoma, FIGO IIIb, G3 p 10:1 ++ / +++
30 Adenocarcinoma, FIGO IV, G3 p 1:1 - / -
31 Solid, pleomorphic adenocarcinoma, FIGO IIIB, G3 p 3:1 + / +++
32 Serous papillary adenocarcinoma, FIGO IIIB, G3 p 1:2 + / +++
33 Serous papillary adenocarcinoma, FIGO IIIc, pN1, G3, carboplatinum/paclitaxel r 1:2.4 - / -
34 Serous papillary adenocarcinoma, FIGO IIIc, pN1, G3, R =1 cm p 1:2 + / *
35 Serous papillary adenocarcinoma, FIGO IIIc, pN1, G3, R >2 cm p 1:1 + / *
36 Solid adenocarcinoma, FIGO IV, pT3c, pNx, G3, p 2:1 ++ / *
37 Serous papillary adenocarcinoma, FIGO IIIc, pN1, G3, R <1 cm p 5:1 - / *
38 Serous papillary adenocarcinoma, FIGO IIIc, pN1, G1, R <1 cm p 1:2 + / *
39 Serous papillary adenocarcinoma, FIGO IIIc, pNx, G3, R <1 cm p 2:1 ++ / ++++
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Specimen preparation

Freshly dissected primary ovarian cancer specimens of 10–30 g were finely minced immediately following primary surgery. Depending on the size of tumors, the tissue mince was placed in 30–90 ml Tumor Dissociation Enzyme reagent (TDE), (Koechli et al. 1993) that contained 0.33 units/ml of collagenase A (Roche, Mannheim, Germany), 0.85 units/ml of dispase grade II (Roche), 144 units/ml DNase I (Sigma, Deisenhofen, Germany), and 15% FBS in RPMI-1640. After 2–3 h incubation at 37 °C, the mince was dissociated into a suspension of single cells and multi-cellular aggregates. For removal of larger cell clusters, the mixture was filtered through a 200-µm filter (Eckert, Waldkirch, Germany) and washed twice with DMEM. A Ficoll density gradient centrifugation was then performed to reduce erythrocyte contamination. The cell suspension from primary tumor specimens was washed twice and resuspended with DMEM containing 10% heat-inactivated FBS to a working concentration of 5×105 cells/ml.

Antibodies

MT201, an Ep-CAM-specific antibody of the human IgG1 isotype was constructed and produced as previously described (Naundorf et al. 2002). Tumor cells expressing Ep-CAM were detected by using the high-affinity antibody anti-Ep-CAM murine monoclonal antibody 3B10 (Micromet, Martinsried, Germany). For FACS analysis, 3B10 was directly labeled with fluorescein-NHS from Sigma. One milligram 3B10 was dialysed against boric buffer (0.05 M boric acid, 0.1 M NaCl, pH 8.3) at room temperature for 3 h, and then incubated with 80 µg fluorescein-NHS, which had been dissolved in DMSO. After 60 min labeling at room temperature, 3B10-FITC was dialysed against PBS overnight at 4 °C. The amount of blood-borne cells in tumor samples was determined by flow cytometry using phycoerythrin (PE-) labeled CD45 antibodies (Biosource, Camarillo, Calif., USA) detecting all cells of hematopoietic origin.

CA-125 is a frequently used and established tumor marker for ovarian cancer (Menon et al. 2000; Gemer et al. 2001). For detection of CA-125 expressing tumor cells, anti-CA-125 (DAKO) labeled with Biotin-LC-LC-NHS (Pierce, Rockford, Ill., USA) and followed Streptavidin-PE (Beckman Coulter, Krefeld, Germany) staining were used. The labeling of anti-CA-125 with biotin was performed according to the manufacturer's specifications.

The specificity of MT201 elimination of Ep-CAM-positive tumor cells was studied by comparing the activity of the human IgG1 MT201 with that of the isotype control rituximab (Roche), a human/murine chimeric IgG1 antibody against the CD20 antigen expressed on B cells (Grillo-Lopez et al. 2000).

Cytotoxicity assay

For assaying specific cytotoxicity, primary tumor cell cultures were seeded at a density of 1×105/ml in 96-well flat-bottom microtiter plates (Costar 3595, Corning, N.Y., USA). MT201 was used at concentrations of 1–10,000 ng/ml diluted in cell culture medium. Plates were then incubated for 1–4 days at 37 °C in a humidified atmosphere of 5% CO2/95% air. New medium was added at the second day.

Lysis of Ep-CAM-positive tumor cells in primary tumor samples was analyzed and quantified by flow cytometry using a FACScan cytometer and the CellQuest analysis program (Beckton Dickinson, Heidelberg, Germany). For FACS analysis, 2×105 cells from primary tumor cell cultures were simultaneously incubated with 100 ng fluorescein-NHS-labeled 3B10 and 10 µl PE-labeled CD45 antibody for 15 min in FACS buffer (PBS/1% FBS) and then washed twice in FACS buffer. In another procedure, the incubation of cells with PE-labeled CD45 was replaced by 100 ng biotin-labeled CA-125 and followed by another 15 min incubation with 25 ng PE-labeled streptavidin. In all cases, 1 µg/ml propidium iodide was added before performing flow cytometry. Effector:target cell ratios (E:T ratios) in each sample were determined as the ratio of CD45- to Ep-CAM-positive cells. The extent of tumor cell depletion was determined by the percent decrease of Ep-CAM-positive tumor cells after gating the live Ep-CAM-positive cells (Ep-CAM+ and PI−) or the double-labeled Ep-CAM- and CA-125-positive tumor cells. Non-specific lysis was determined in all experiment shown. It provided the basis for calculating the specific cell lysis shown in the figures. Non-specific lysis seen in the absence of monoclonal antibody was typically in the order of 5%. In the case of OvCAR-3 cells, PBMCs from healthy human donors were added at E:T ratios of 10:1 and 1:1.

Isolation of Ep-CAM-positive cells and cytology

To isolate Ep-CAM-positive cells from cell suspensions of freshly dissected ovarian tumors, magnetic Dynabeads (M-456 rat anti-mouse IgG1) from Dynal (Oslo, Norway) were used according to the manufacturer's guidelines. Briefly, 1 µg anti-Ep-CAM mouse monoclonal antibody 3B10 was incubated with 1×108 Dynabeads for 30 min at 4 °C, washed with PBS with 0.1% FBS three times and resuspended to a concentration of 1×107 beads/ml. Cells (2×106) from ovarian cancer tissue, which was enzymatically dissociated as described above, were incubated with 1 ml Dynabeads coated with 3B10 for 30 min at 4 °C, and subsequently washed twice. The purified Ep-CAM cells were then prepared for cytospin and stained with Giemsa dye for cytological examination.

Results

ADCC by MT201 against OvCAR-3 cells

To evaluate the anti-tumor efficacy of MT201, we first investigated the effect of the human monoclonal antibody on the ovarian tumor cell line OvCAR-3. The vast majority of OvCAR-3 cells expressed both Ep-CAM and CA-125 (Fig. 1A,B). In cocultures with PBMC, double-labeling of OvCAR-3 cells allowed us to separate a double-stained cell population (in gate R1) from the background of CD45-positive effector cells. The reduction of this propidium iodide-negative (i.e., live) cell population in gate R1 provided for an assay to monitor by flow cytometry the ADCC of MT201.

Fig. 1A–C.

Fig. 1A–C.

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The effect of MT201 on the vitality of Ep-CAM/CA-125 double-labeled OvCAR-3 cells in a FACS-based cytotoxicity assay. OvCAR-3 cells (gate R1) were mixed with human PBMC at a ratio of 1:1 and cultured in the A absence or B presence of the indicated concentrations of MT201; C Quantification of time- and dose-dependent specific cell lysis by MT201

MT201 at concentrations ranging from 10–1,000 ng/ml effectively eliminated OvCAR-3 tumor cells in coculture with PBMC during the 4-d incubation period at an E:T ratio of 1:1 (Fig. 1B). The lysis of tumor cells was time- and dose-dependent (Fig. 1C). After 4 days, MT201 depleted more than 90% of tumor cells at a concentration of 1 µg/ml. Half maximal ADCC was observed at a concentration of <10 ng/ml. Specific cell lysis was determined by subtraction of cell lysis observed in the absence of MT201.

The effect of MT201 on specific cell lysis of OvCAR-3 cells under suboptimal conditions could be significantly enhanced by increasing the E:T ratio. As shown in Fig. 2, there was no significant tumor cell lysis after a 16-h incubation at an E:T ratio of 1:1. Increased specific cell lysis by MT201 could, however, be observed at an E:T ratio of 5:1 with forty percent cell lysis reached by 10 µg/ml MT201. At an E:T ratio of 10:1, the specific depletion of tumor cells was further enhanced. Only 1 ng/ml MT201 could now induce 55% lysis of tumor cells. Eighty percent tumor cell lysis was obtained at the highest concentration of 10 µg/ml.

Fig. 2.

Fig. 2.

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The effect of E:T ratio on the cytotoxic activity of MT201 against OvCAR-3 cells. PBMC were present at E:T ratios referred to CD45-positive cells of 1:1, 5:1, and 10:1. MT201 was tested at the indicated concentrations. After 16-h incubation, cells were labeled with anti-Ep-CAM 3B10-FITC, CD45-PE, and propidium iodide (PI) and viable cells quantitated by flow cytometry

Ex vivo lysis of primary tumor cells by MT201

We further performed ex vivo studies on the specific cytotoxicity of MT201 against Ep-CAM-positive tumor cells present in freshly dissected peritoneal metastatic tissue of patients with primary ovarian cancer.

The percentage of tumor cells expressing Ep-CAM was determined by flow cytometry. As can be seen in Fig. 3A freshly isolated cells prepared from a primary ovarian cancer contained a significant population of propidium iodide-negative- Ep-CAM cells (Ep-CAM+ and PI−) as highlighted in gate R1. Of note, the majority of Ep-CAM-positive cells in tumor samples also expressed the tumor-associated antigen CA-125 (Fig. 3B, gate R2). Only very few cells were detected that were Ep-CAM- or CA-125 single-labeled. Ep-CAM-positive cells isolated from primary ovarian tumors by anti-Ep-CAM antibody 3B10-conjugated magnetic beads and subsequently stained with Giemsa showed that essentially all isolated Ep-CAM-positive cells had the morphology of tumor cells, such as enlarged nuclei (Fig. 3C).

Fig. 3A,B.

Fig. 3A,B.

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The proportion of Ep-CAM-positive cells among primary tumor cells from ovarian cancer tissue. A Flow cytometry employing Ep-CAM labeling combined with propidium iodide uptake of cells or B Ep-CAM/CA-125 double-labeling. Gate R1 highlights live Ep-CAM-positive cells and Gate R2 Ep-CAM-/CA-125 double-positive cells; C Cytological examination of Ep-CAM-positive cells isolated from tumor tissue by anti-Ep-CAM magnetic beads (arrows). A microphotograph of Giemsa-stained cells is shown. The bar represents a length of 20 µm

The effect of MT201 on the viability of Ep-CAM-positive cells in disseminated tumor tissue from 21 patients with ovarian cancer were analyzed by the flow cytometry-based ADCC assay. The anti-Ep-CAM antibody 3B10-FITC, anti-CD45-PE, and propidium iodide were simultaneously applied in the assay, in order to mark the immune cells (CD45+ and PI−) and alive Ep-CAM-positive tumor cells (Ep-CAM+ and PI−). Specific cytotoxicity of MT201 was investigated at antibody concentrations ranging from 1 to 10, 000 ng/ml and for incubation times of 16 h and 30 h.

FACS analysis from a selected patient specimens showed that the population of alive Ep-CAM-positive cells seen in gate R1 was reduced with increasing concentrations of MT201 after 30 h of incubation (Fig. 4A). Dose-response analyses for two patient specimens (nos. 32 and 39) showed that MT201 efficiently lysed Ep-CAM-positive cells after 30 h within the complex cell mixture derived from primary tumor material (Fig. 4B,C). Reduced cell lysis was observed after 16 h. At a concentration of 1 µg/ml MT201, between 50% and 80% specific cell lysis was observed after 30 h of incubation. Doses as low as 1 ng/ml already showed a significantly enhanced tumor cell lysis compared to untreated controls. Of note, no extra immune cells were added, suggesting that the endogenous tumor-resident effector cells mediated ADCC by MT201.

Fig. 4A–C.

Fig. 4A–C.

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Lysis of Ep-CAM-positive cells by MT201 in tumor tissue samples from selected patients. A FACS scans of disseminated cells from tumor sample of patient no. 32. Cells were treated by different concentrations of MT201 for 30 h. Gate R1 highlights a population of viable Ep-CAM-positive cells; B,C Dose-response analyses of specific cell lysis by MT201 in two ovarian cancer patient samples (nos. 32 and 39). Samples had E:T (CD45:Ep-CAM) ratios of 1:2 and 2:1, respectively. MT201 concentrations and incubation periods are indicated. Data are from a FACS-based cytotoxicity assay

The cytotoxic activity of MT201 was studied with a total of 21 tumor samples, as summarized in Table 1. The ratio of endogenous CD45-positive immune cells to Ep-CAM-positive cells in these sample ranged widely from 1:2.4 to 12:1. Ep-CAM-positive cells derived from 17 patients were sensitive towards MT201 at concentrations between 1 and 10,000 ng/ml showing an overall response rate of 81%. Sixteen out of 21 patient samples (76%) responded to MT201 already after 16 h of treatment. In seven cases (patient nos. 17, 23, 25, 27, 31, 32, and 39), we could observe a further increased cell lysis after 30 h of incubation with MT201. There was no obvious correlation between the responsiveness of tumor cells to MT201 and the abundance of CD45+ immune cells or the histological tumor grading of respective patients.

Where tested, tumor specimens contained immune cell subpopulations that were positive for CD4, CD8, CD56, TCR alpha/beta, TCR gamma/delta, NK cell markers, and CD45RO or CD45RA (data not shown). As expected, tumor samples showed considerable variation with respect to immune cell composition (data not shown). The limited amount of tumor tissue did not allow us to routinely analyze immune cell phenotypes for all specimen.

In order to determine the specificity of MT201-mediated depletion of Ep-CAM-positive tumor cells within disseminated tumor tissue, we compared the activity of MT201 with that of rituximab, an CD20-specific IgG1 antibody. Figure 5 shows the result with the tumor sample from patient no. 39. At an E:T ratio of 2:1, only MT201 but not equivalent concentrations of the human (chimeric) IgG1 Rituximab showed a significant lysis of tumor cells.

Fig. 5.

Fig. 5.

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Specificity of MT201 anti-tumor activity for Ep-CAM. A tumor sample from ovarian cancer patient no. 39 was used. The ratio of endogenous CD45-positive immune cells to Ep-CAM-positive cells was 2:1. Cell cultures were treated with either MT201 or the chimeric IgG1 monoclonal antibody rituximab for 30 h. Concentrations of MT201 and rituximab tested are indicated. Data are from a FACS-based cytotoxicity assay

Discussion

Our study describes the antitumor activity of the fully human, recombinant anti-Ep-CAM monoclonal antibody MT201 using disseminated primary tumor samples from ovarian cancer patients. MT201 was developed as human IgG1 to achieve a higher cytotoxic potential and lower immunogenicity than the validated murine IgG2a antibody edrecolomab, while preserving the broad therapeutic window of edrecolomab. In the present study, we employed a novel approach to evaluate the anti-tumor potential of a monoclonal antibody. Metastatic tumor masses were removed from patients, disseminated, and cell mixtures from tumor cultured in vitro. Dissemination was necessary to enable cell culture, single out, and characterize tumor cells and to subsequently determine the fate of tumor cells in a flow cytometry-based assay. Otherwise, the cultured tumor-derived cells were not drastically changed in their cellular composition except for removal of clumps >200 µm and of erythrocytes. Most importantly, ADCC reactions of MT201 with this material entirely relied on the endogenous, autologous immune cells of patients. Their percentage was estimated by FACS-based determination of CD45-positive cells. This clearly underestimated the efficacy of MT201 because E:T ratios considered all CD45-positive blood-borne cells and not just the fraction of Fcγ receptor-positive effector cells capable of ADCC.

We have observed a potent MT201-mediated specific cytotoxicity with the majority of tumor samples. This cytotoxicity was largely mediated by ADCC and not by CDC because tumor-derived cells were freed by the washing of complement, and the use of heat-inactivated FBS for the cell culture. We could not observe that MT201 had any cytotoxic activity in the absence of effector cells or complement on various Ep-CAM-positive tumor lines (data not shown)(Naundorf et al. 2002). Hence, our data suggest that MT201 has a high anti-tumor activity that is primarily mediated via ADCC by the tumor-resident effector cells. Moreover, the data suggest that other cell types and their products derived from the tumor tissue did not strongly affect recruitment and activation of effector cells by MT201.

The degree of tumor cell lysis seen in in vitro reactions with tumor samples was generally not sufficient for a complete elimination of Ep-CAM-positive cells. Exhaustion of effector cells, accumulation of factors inhibiting immune cell activity, degradation of MT201 or too short incubation times may be possible explanations. In vivo, the efficacy of MT201 could be considerably higher because there is a fresh supply of immune cells from blood, the removal of inhibitory factors by diffusion, the attraction of local immune cells by chemokines, the typically long half-life of IgG1, and the supply of fresh antibody from the circulation and by repeated infusion. In addition, CDC by MT201 is likely to provide in vivo an additional cytotoxic effector mechanism on top of ADCC. There are also factors that could potentially reduce MT201 activity in vivo. One is impaired penetration of MT201 into tumors due to high interstitial pressure and large antibody size. The other is the presence of excess human IgG potentially competing with MT201 for Fcγ-receptor-binding on effector cell as observed in vitro (Naundorf et al. 2002).

The high response rate of ovarian tumor samples to MT201-mediated cytotoxicity was surprising. We could not see an obvious correlation between tumor cell lysis and the ratio of CD45+:Ep-CAM+ cells. In two cases (patient samples nos. 23 and 24), the tumor cells showed almost no response to MT201 despite abundant CD45+ cells. On the other hand, some samples (e.g., nos. 20 and 32) did effectively respond to MT201 even at very unfavorable ratios of CD45+:Ep-CAM+ cells. An explanation could be that the content of Fcγ receptor-positive effector cells among CD45+ cells was different in the individual patient samples. Likewise, vitality and activity of immune cells could have been different. We conclude from the high response rate that the various cell types in ovarian cancer tissue, such as tumor cells, fibroblasts, stromal cells, and endothelial cells, and their extracellular products did not strongly interfere with ADCC of MT201.

The fully human nature of MT201 is expected to allow for a repeated administration of the antibody without the kind of neutralization observed for edrecolomab by a human-anti-mouse antibody response (Khazaeli et al. 1994; Welt 1998). Repeated treatment with MT201 in combination with the long half-life of human IgG1 may impose a continuous immune surveillance over metastatic tumor cells and improve the chance of the antibody to penetrate solid tumors and extravascular compartments. Moreover, the human nature of MT201 may allow for a better interaction with Fcγ receptor-positive human immune effector cells translating into higher efficacy of ADCC (Naundorf et al. 2002).

The evaluation of therapeutic anti-cancer antibodies at the pre-clinical level typically relies on studying established human tumor cell lines. Those are usually investigated for their elimination by antibodies in vitro via ADCC and CDC (complement-dependent cytotoxicity) and, in vivo, as xenotransplants in nude or severe combined immunodeficiency syndrome (SCID) mouse models (Masucci et al. 1998; Nishihara et al. 2000). The homogeneity of tumors derived from rapidly growing cancer cell lines in mice do not necessarily reflect the heterogeneity and cellular complexity of slowly growing natural tumors. For that matter, the potential of the monoclonal antibodies tested in mouse models are frequently overestimated. As consequence, response rates determined in animal models are usually much higher than subsequently observed in patients. Moreover, animal models typically use cell lines for tumor establishment that are most susceptible to the antibody in vitro. In the present study no such selection occurred. Instead, the primary tumor tissue from a sizable number of patients was examined for elimination of tumor cells through endogenous effector cells. We expect that this experimental approach may have a higher predictive value for clinical outcome than animal models using cell lines.

MT201 may have an advantage compared to other monoclonal antibodies tested in ovarian cancer such as M0v18, HER2-antibodies, and anti-idiotypic antibodies. Bookmaet al. showed an overall response rate of only 7.3% after single-agent monoclonal antibody therapy targeting HER2/neu (trastuzumab) in ovarian and primary peritoneal carcinoma. The clinical value of single-agent trastuzumab in recurrent ovarian cancer is limited by the low frequency of HER2 overexpression and low rate of objective response among patients with HER2 overexpression (Bookman et al. 2003). Schlebusch et al. presented data with a murine anti-idiotypic antibody (ACA 125) imitating the tumor-associated antigen CA 125, which can be detected in about 80% of ovarian carcinomas. In vitro CDC as well as ADCC was observed by ACA 125, but further investigations in vivo are necessary for determination of its efficacy (Schlebusch et al. 1995). M0v18 is a chimeric monoclonal that binds the membrane folate receptor expressed on ovarian carcinoma cells. The effect of M0v18 was studied in 14 patients after intraperitoneal and intravenous application of the antibody. The uptake in solid tumor tissue in ovarian cancer patients operated 6 days post-injection was not significantly different for both routes (van Zanten-Przybysz et al. 2001). Future clinical trials in ovarian cancer patients will determine efficacy, response rates, and optimal application of monoclonal antibody MT201.

Acknowledgments

The authors would like to thank Mrs. D. Egner and Mrs. M. Fileki for their technical assistance.

Abbreviations

ADCC

Antibody-dependent cellular cytotoxicity

CDC

Complement-dependent cytotoxicity

Ep-CAM

Epithelial cell adhesion molecule

E:T ratio

Effector to target ratio

FACS

Fluorescence-activated cell sorting

FBS

Fetal bovine serum

mAb

Monoclonal antibodies

PBMC

Peripheral blood mononuclear cells

PE

Phycoerythrin

Footnotes

The first two authors contributed equally to this study.

References

  1. Abdullah N, Greenman J, Pimenidou A, Topping KP, Monson JRT (1999) The role of monocytes and natural killer cells in mediating antibody-dependent lysis of colorectal tumour cells. Cancer Immunol Immunother 48:517–524 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Averette HE, Janicek MF, Menck HR (1995) The National Cancer Data Base report on ovarian cancer. American College of Surgeons Commission on Cancer and the American Cancer Society. Cancer 76:1096–1103 [DOI] [PubMed] [Google Scholar]
  3. Balzar M, Winter ML, de Boer CJ, Litvinov SV (1999) The biology of the 17–1A antigen (Ep-CAM). J Mol Med 77:699–712 [DOI] [PubMed] [Google Scholar]
  4. Bookman MA, Darcy KM, Clarke-Pearson D, Boothby RA, Horowi IR (2003) Evaluation of monoclonal humanized anti-HER2 antibody, trastuzumab, in patients with recurrent or refractory ovarian or primary peritoneal carcinoma with overexpression of HER2: a phase II trial of the Gynecologic Oncology Group. J Clin Oncol 21:283–290 [DOI] [PubMed] [Google Scholar]
  5. Fields ALA, Keller AM, Schwartzberg L, Bernard S, Pazdur R, Kardinal C, Cohen A, Schultz J, Eisenberg P, Barber L, Edmundson S, Wissel P (2002) Edrecolomab (17–1A antibody) (EDR) in combination with 5-fluorouracil (FU) based chemotherapy in the adjuvant treatment of stage III colon cancer: results of a randomised north american phase III study. ASCO [abstr 508]
  6. Gemer O, Segal S, Kopmar A (2001) Preoperative CA-125 level as a predictor of non-optimal cytoreduction of advanced epithelial ovarian cancer. Acta Obstet Gynecol Scand 80:583–586 [PubMed] [Google Scholar]
  7. Grillo-Lopez AJ, Write CA, Dallaire BK, Varns CL, Shen CD, Wei A, Leonard JE, McClure A, Weaver R, Cairelli, R, Rosenberg J (2000) Rituximab: the first monoclonal antibody approved for the treatment of lymphoma. Curr Pharm Biotechnol 1:1–9 [DOI] [PubMed] [Google Scholar]
  8. Khazaeli MB, Conry RM, LoBuglio AF (1994) Human immune response to monoclonal antibodies. J Immunother 15:42–52 [DOI] [PubMed] [Google Scholar]
  9. Koechli OR, Avner BP, Sevin B-U, Avner B, Perras JP, Robinson DS, Averette HE (1993) Application of the adenosine triphosphate-cell viability assay in human breast cancer chemosensitivity testing: a report on the first results. J Surg Oncol 54:119–125 [DOI] [PubMed] [Google Scholar]
  10. Masucci G, Lindemalm C, Frodin JE, Hagstrom B, Mellstedt, H (1988) Effect of human blood mononuclear cell populations in antibody dependent cellular cytotoxicity (ADCC) using two murine (CO17–1A and Br55–2) and one chimeric (17–1A) monoclonal antibodies against a human colorectal carcinoma cell line (SW948). Hybridoma 7:429–440 [DOI] [PubMed] [Google Scholar]
  11. McGuire WP, Hoskins WJ, Brady MF, Kucera PR, Partridge EE, Look KY, Clarke-Pearson DL, Davidson M (1996) Cyclophosphamide and cisplatin versus paclitaxel and cisplatin: a phase III randomized trial in patients with suboptimal stage III/IV ovarian cancer (from the Gynecologic Oncology Group). Semin Oncol 23:40–47 [PubMed] [Google Scholar]
  12. McLaughlin PM, Harmsen MC, Dokter WH, Kroesen BJ, van der Mohlen H, Brinker MG, Hollema H, Ruiters MH, Buys CH, de Leij LF (2000) The epithelial glycoprotein 2 (EGP-2) promoter-driven epithelial-specific expression of EGP-2 in transgenic mice: a model to study carcinoma-directed immunotherapy. Cancer Res 15:4105–4111 [PubMed] [Google Scholar]
  13. Menon U, Jacobs IJ (2000) Recent developments in ovarian cancer screening. Curr Opin Obstet Gynecol 12:39–42 [DOI] [PubMed] [Google Scholar]
  14. Naundorf S, Preithner S, Mayer P, Lippold S, Wolf A, Hanakam F, Fichtner I, Kufer P, Raum T, Riethmüller G, Baeuerle PA, Dreier T (2002) In vitro and in vivo activity of MT201, a fully human monoclonal antibody for pancarcinoma treatment. Int J Cancer 100:101–110 [DOI] [PubMed] [Google Scholar]
  15. Nishihara T, Sawada T, Yamamoto A, Yamashita Y, Ho JJ, Kim YS, Chung KH (2000) Antibody-dependent cytotoxicity mediated by chimeric monoclonal antibody Nd2 and experimental immunotherapy for pancreatic cancer. Jpn J Cancer Res 91:817–824 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Parker SL, Tong T, Bolden S, Wingo PA (1997) Cancer statistics. CA Cancer J Clin 46:5–27 [DOI] [PubMed] [Google Scholar]
  17. Punt CJ, Nagy A, Douillard JY, Figer A, Skovsgaard T, Monson J, Barone C, Fountzilas G, Riess H, Moylan E, Jones D, Dethling J, Colman J, Coward L, MacGregor S (2002) Edrecolomab alone or in combination with fluorouracil and folinic acid in the adjuvant treatment of stage III colon cancer: a randomised study. Lancet 360:671–677 [DOI] [PubMed] [Google Scholar]
  18. Raum T, Gruber R, Riethmüller G, Kufer P (2001) Anti-self antibodies selected from a human IgD heavy chain repertoire: a novel approach to generate therapeutic human antibodies against tumor-associated differentiation antigens. Cancer Immunol Immunother 50:141–150 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Riethmüller G, Schneider-Gädicke E, Schlimok G, Schmiegel W, Raab R, Höffken K, Gruber R, Pichlmaier H, Hirche H, Pichlmayr et al (1994) Randomised trial of monoclonal antibody for adjuvant terapy of resected Dukes'C colorectal carcinoma. Lancet 343:1177–1183 [DOI] [PubMed] [Google Scholar]
  20. Riethmüller G, Holz E, Schlimok G, Schmiegel W, Raab R, Hoffken K, Gruber R, Funke I, Pichlmaier H, Hirche H, Buggisch P, Witte J, Pichlmayr R (1998) Monoclonal antibody therapy for resected Dukes'C coloretal cancer: seven-year outcome of a multicenter randomized trial. J Clin Oncol 16:1788–1794 [DOI] [PubMed] [Google Scholar]
  21. Schlebusch H, Wagner U, Grunn U, Schultes B (1995) A monoclonal antiidiotypic antibody ACA 125 mimicking the tumor-associated antigen CA 125 for immunotherapy of ovarian cancer. Hybridoma 14:167–174 [DOI] [PubMed] [Google Scholar]
  22. Sears HF, Atkinson B, Mattis J, Ernst C, Herlyn D, Steplewski Z, Hayry P, Koprowski H (1982) Phase-I clinical trial of monoclonal antibody in treatment of gastrointestinal tumours. Lancet 1:762–765 [DOI] [PubMed] [Google Scholar]
  23. Welt S (1998) Antibodies 1998: Molecular, cellular and clinical aspects. Cancer Res Inst 17 [Google Scholar]
  24. Young R, Perez C, Hoskins W (1993) Cancer of the ovary. In: DeVita VT, Hellmann S, Rosenberg SA (eds) Cancer: principles and practice of oncology. Lippincott, Philadephia
  25. Van Zanten-Przybyz I, Molthoff CF, Roos JC, Verheijen RH, van Hof A, Buist MR, Prinssen HM, den Hollander W, Kenemans P (2001) Influence of the route of administration on targeting of ovarian cancer with the chimeric monoclonal antibody M0V18: i.v. vs. i.p. Int J Cancer 92:106–114 [PubMed] [Google Scholar]

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