Journal of Histochemistry and Cytochemistry, Vol. 49, 911-918, July 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Lysis of Prostate Carcinoma Cells by Trifunctional Bispecific Antibodies ({alpha}EpCAM x {alpha}CD3)

Rainer Riesenberga, Alexander Buchnera, Heike Pohlaa, and Horst Lindhoferb
a Laboratory for Tumorimmunology, Department of Urology, Ludwig-Maximilians-University, Klinikum Grosshadern, Munich, Germany
b Clinical Cooperation group "Bispecific Antibodies" at the Department of Otorhinolaryngology, Ludwig-Maximilians-University, and the Institute of Clinical Molecular Biology, GSF–National Research Center for Environment and Health, Munich, Germany

Correspondence to: Rainer Riesenberg, Labor für Tumorimmunologie, Urologische Klinik, Klinikum Grosshadern, Marchioninistr. 23, D-81377 Munich, Germany. E-mail: riesenberg@life.med.uni-muenchen.de


  Summary
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Materials and Methods
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Discussion
Literature Cited

Bispecific monoclonal antibodies (bsAbs) are a promising immunotherapeutic option for treatment of cancer, especially in situations of minimal residual disease. The combination of an anti-CD3 and anti-tumor-associated antigen antibody redirects cytotoxic T-lymphocytes towards malignant cells. Using a trifunctional bispecific antibody against EpCAM x CD3, that additionally activates Fc{gamma}R+ accessory cells via its Fc region, we investigated the interaction between three EpCAM+ prostate carcinoma cell lines and peripheral blood mononuclear cells (PBMCs) of healthy donors and patients with prostate carcinoma (PC). Visualization was performed by double immunocytochemical methods and computerized sequential video microscopy. Tumor cells and PBMCs supplemented with {alpha}EpCAM x {alpha}CD3 in 16-well chamber slides resulted in lysis of tumor cells within 1–3 days without any differences between patient and healthy donor PBMCs. The characteristic necrotic way of tumor cell killing (rounding, swelling, disrupting) could be observed in computerized sequences of video frames. Simultaneously, we could not reveal any form of apoptotic signal using three different apoptotic markers (TUNEL, M30 cyto death, anti-active caspase 3). Within the first 48 hr we observed typical PBMC cluster formation with increasing cell proliferation. PBMCs surrounding the tumor cells were not dominated by CD4+, CD8+, or CD14+ cells. Lymphocytes with pore-forming perforin proteins concentrated towards the tumor target cells. Our combination of double immunocytochemical and computerized video microscopic techniques may serve as an important improvement of validity of cell–cell interaction experiments using in vitro models. (J Histochem Cytochem 49:911–917, 2001)

Key Words: prostate carcinoma, bispecific antibody, double immunocytochemistry, computerized sequential, microscopy


  Introduction
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Introduction
Materials and Methods
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EVEN after successful treatment of cancer using conventional methods, there is still a high risk for recurrent disease. The main problem in treatment today is not the primary tumor itself but the disseminated and still viable tumor cells. These can migrate within the patient's body and cause, years later, untreatable metastases and/or recurrence of the original tumor. It is therefore important to ensure that any remaining tumor cells are specifically and efficiently eliminated to prevent relapse of the disease.

As animal studies have demonstrated, trifunctional bispecific antibodies (bsAbs) represent a particularly effective weapon in the elimination of residual tumor cells (Lindhofer et al. 1996 ). This has been made possible by a mode of action that represents a further development of the conventional bispecific antibodies. Trifunctional bsAbs are constructed with the intent of attracting not only T-cells but at the same time accessory cells to the tumor cells, where they are activated. The target cells are thus simultaneously subjected to various destructive processes and consequently die (Zeidler et al. 1999 ). A major benefit of this technique is that tumor material is also internalized, processed, and presented to the immune system on the surface of professional antigen-presenting cells (Zeidler et al. 2000 ).

The EpCAM antigen, a surface glycoprotein expressed by cells of simple epithelia, has been shown to be a rewarding target for monoclonal antibody therapy against disseminated tumor cells in patients with minimal residual colorectal cancer (Riethmuller et al. 1994 ). The anti-EpCAM x anti-CD3 bispecific antibody used in our investigations recognizes EpCAM on epithelial tumor cells, CD3 complexes on T-lymphocytes, and activates Fc{gamma}R+ accessory cells (e.g., monocytes, macrophages, dendritic cells) via its Fc region (Zeidler et al. 1999 ). By means of formation of this tri-cell complex, the immune cells are brought into direct contact with the tumor cells and are specifically activated which benefits, e.g., in the context of ex vivo purging of stem cell preparations (Lindhofer et al. 1997 ), where systemic side effects do not have particular importance.

We could directly demonstrate the tissue-specific origin of cytokeratin-positive disseminated tumor cells in bone marrow of patients with primary adenocarcinomas of the prostate using MAbs recognizing prostate-specific antigen (PSA) (Riesenberg et al. 1993 ). In this study we demonstrate for the first time the lysis of prostate carcinoma (PC) cells in mixed populations of tumor cells and PBMCs after treatment with trifunctional bispecific antibodies using double immunocytochemistry methods and video microscopic techniques.


  Materials and Methods
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Materials and Methods
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Production of Bispecific Antibodies
The TPBS01 quadroma was produced as previously described (Lindhofer et al. 1995 ). The following hybridomas have been used: 26II6 (rat IgG2b, anti-CD3, kindly provided by R. Schuh, GSF–National Research Center for Environment and Health, Munich, Germany) and HO-3 (mouse IgG2a, anti-EpCAM). The bispecific antibody anti-EpCAM x anti-CD3 was kindly provided by Trion Pharma (Munich, Germany).

Cell Lines
The established EpCAM+ prostate tumor cell lines PC3, DU145, and LNCaP, as well as the EpCAM- bladder carcinoma cell line J82 (obtained from American Type Culture Collection; Manassas, VA), were grown in RPMI 1640 (Biochrom; Berlin, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 4 mM L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin (all GIBCO/BRL; Eggenstein, Germany). Cells were maintained at 37C in a humidified atmosphere of 5% carbon dioxide/95% air as a subconfluent monolayer in 260 ml/83 cm2 tissue flasks (Nunc; Karlsruhe, Germany).

Experimental Model
Exponentially proliferating tumor cells were harvested with 0.05% trypsin/0.02% EDTA (GIBCO/BRL; Karlsruhe, Germany). Unstimulated human PBMCs from heparinized blood of healthy donors and patients with PC were isolated by density centrifugation through Ficoll–Hypaque (Pharmacia, Uppsala, Sweden; Loos and Roos 1974 ). Mixed populations of tumor cells (4000 cells/well) and PBMCs (1 x 105 cells/well) were set up in 16-well chamber slides (Nunc) in complete RPMI 1640 medium (300 ml/well) supplemented with 30 ng/well bsAbs and incubated up to 10 days. In parallel, control cultures were seeded (without bsAbs or tumor cells, respectively) and treated in the same way. At a given time, medium was carefully removed. After air-drying, slides were either stained immediately or stored at -80C. The growth chambers on this standard microscope slide can be removed when staining is complete.

Computerized Sequential Video Microscopy
Computerized storage of video frames allows the observation of movements of cultured cells by quick-time video clips using special video software. For this purpose, chambers were labeled by a transparency to delay variations in pH and the slide was positioned on the stage of an inverted microscope (Axiovert 35; Zeiss, Jena, Germany) equipped with a stage heater device that maintains a temperature of 37C within the individual wells.

A video camera (Tricam SL; Storz, Tuttlingen, Germany) was mounted in the optical path of the microscope. Capturing of video frames was performed using a personal computer equipped with specialized hard- and software (mfg-board, Optimas 6.2; Stemmer Imaging, Puchheim, Germany). Pictures of the cell culture chamber were taken at 30-sec intervals and stored to the hard disk in the JPEG format over the full experimental period (up to 3 days). Quick-time video clips in MPEG-2 format were created from the JPEG file sequences using video editing software (Media Studio Pro 6.0; Ulead Systems, Braunschweig, Germany).

Double Immunocytochemistry
Slides were thawed, air-dried, fixed in 1% formaldehyde for 10 min, and washed three times in PBS (0.01 M, pH 7.4). Slides were transferred to a moist chamber and preparations were horizontally incubated for 20 min in PBS containing 10% AB serum (Biotest; Dreieich, Germany) to reduce nonspecific binding. Intact mouse monoclonal antibodies (anti-perforin, anti-Ki67), antibody–HRP conjugates (anti-CD3), or biotinylated antibodies (anti-CD4, anti-CD8, anti-CD14) were applied for 45 min followed by an incubation with rabbit anti-mouse antibody HRP-conjugated (DAKO, Glostrup, Denmark; diluted 1:100 in PBS) or streptavidin–HRP conjugate respectively (DAKO; diluted 1:150) for 30 min (antibodies are summarized in Table 1). Each incubation step was interrupted by thoroughly washing the slides three times in PBS to ensure complete removal of unbound antibodies. Visualization of specifically bound antibodies or streptavidin–HRP conjugate was done in a solution of 16 mg 3-amino-9-ethylcarbazole (AEC) in 4 ml dimethyl-formamide (Sigma; St Louis, MO) mixed with 46 ml sodium acetate buffer (pH 5.2) and 60 µl H2O2 (30%) for 15 min. AEC forms an insoluble red–brown reaction product.


 
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Table 1. Primary antibodiesa

The second part of double staining was initiated with the incubation of samples in PBS/5% mouse serum (DAKO) for 20 min to avoid binding of antibodies to murine antigens. Samples were then incubated for 45 min with the alkaline phosphatase-conjugated F(ab)2 fragment of the monoclonal anti-pan-cytokeratin antibody A45-B/B3 (Micromet; Munich, Germany) to identify cytoskeletal cytokeratins of epithelial cells. The alkaline phosphatase reaction was developed by a substrate–chromogen solution of 5-bromo-4-chloro-3-indoxyl phosphate and nitroblue tetrazolium chloride (BCIP/NBT; DAKO), which yields a blue–purple reaction product at the site of the target antigen.

Detection of Apoptosis
TUNEL. The characteristic DNA fragmentation in apoptotic cells is first produced by endonucleolytic degradation of higher-order chromatin structural organization. The ultimate DNA fragments are multimers of about 180 bp nucleosomal units. These DNA strand breaks can be detected by enzymatically labeling the free 3'-OH termini using the enzyme terminal deoxynucleotidyl transferase (TdT), which forms a polymeric tail using the principle of the TUNEL assay (TdT-mediated dUTP nick end-labeling). We applied the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Intergen; Oxford, UK) according to the manufacturer's instructions. DNA fragments that have been labeled with digoxigenin-nucleotides are then allowed to bind an anti-digoxigenin antibody that is conjugated to peroxidase, which can be visualized using AEC as substrate. Negative control was performed by omitting TdT. As a positive control we used camptothecin-treated HL-60 cells.

M30 CytoDeath. Caspases mediate the cleavage of natural intracellular proteins during apoptosis. Cytokeratins, in particular cytokeratin 18, are affected in very early events of apoptosis. We used the M30 CytoDeath monoclonal antibody, recognizing the caspase cleavage product of cytokeratin 18 (Roche, Mannheim, Germany; diluted 1:10 in PBS, Clone M30) which is not detectable in native CK18 of normal cells. In contrast to the TUNEL reaction, this antibody characterizes a very early and definite event during the apoptotic process. The staining procedure was carried out similarly to the first part of the double staining described previously. M30 antibodies were applied for 45 min, followed by an incubation with rabbit anti-mouse HRP-conjugated antibody (DAKO) for 30 min. Visualization of specifically bound antibodies was carried out with the routine protocol for peroxidase staining using AEC.

Active Caspase 3. Caspase 3 is a key protease that is activated during the early stages of apoptosis. Once caspase 3 has been activated, there is no return to normal viability; the program for cell death is irreversibly activated. Slides were treated with biotin-conjugated polyclonal anti-active caspase-3 (Pharmingen, Heidelberg, Germany; diluted 1:50 in PBS) for 45 min, followed by an incubation with streptavidin–HRP conjugate (DAKO; diluted 1:150) for 30 min and peroxidase staining using AEC.


  Results
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Materials and Methods
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To visualize the interactions of CD3+ and CD14+ PBMCs from healthy donors and patients with PC in reaction to the presence of prostate tumor cells and trifunctional bsAb (anti CD3/anti EpCAM), we investigated mixed populations of tumor cells and PBMC (effector:target ratio 25:1; Zeidler et al. 1999 ) in 16-well chamber slides. Thus, it is possible to observe 16 different experiments under comparable terms and conditions with only small volumes of ingredients. The experiments can be stopped at any time, the chambers can be removed from the slides, and the resulting samples can subsequently be stained immunocytochemically.

First of all, we observed the alterations of the two different cell populations at regular intervals. The adherent growing cells of the EpCAM+ cell lines (PC3, DU145, LNCaP) were surrounded by increasing amounts of PBMCs 6 hr after starting the experiment. Within the next 18 hr, several increasing clusters of proliferating lymphocytes could be detected. Simultaneously, the morphology of the tumor cells altered spherically, with remarkable changes in cell volume (Fig 1b). At 24 hr after starting the experiment, most of the tumor cells were eliminated. After a total time of 72 hr no tumor cells could be detected. We could not observe any difference in cell behaviour between PBMCs from healthy donors and those from patients with PC. Mixed cultures without bispecific antibodies displayed neither clusters of PBMCs nor variations in morphology of tumor cells (Fig 1a). Formation of PBMC clusters could be observed in the presence of bispecific antibodies without tumor cells. When tumor cells were allowed to grow adherent for 1 or 2 days and were then incubated with PBMCs and bsAb, they behaved similarly as already described: tumor cells disrupted after cluster formation of PBMCs with a slight time delay due to preceding tumor cell proliferation. In contrast to that, the EpCAM- J82 cells were not affected by bsAb and PBMCs, although we could observe the typical cluster formation of PBMCs. The results of these investigations were confirmed by at least two further replicates.



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Figure 1. (a) May–Gruenwald–Giemsa staining of PC3 tumor cells and PBMCs (arrowheads) after 24-hr culturing of PBMCs and PC3 without BsAk. (b) May–Gruenwald–Giemsa staining of PC3 tumor cells and PBMCs after 24-hr culturing of PBMCs and PC3 supplemented with BsAb. Morphology of tumor cells changes considerably in size and volume (arrows), whereas PBMCs proliferate. (c) CD3+ T-cells (brown) after 10-day culturing of PBMCs and PC3 tumor cells (black) without BsAb. Both cell populations do not exhibit any cell–cell interaction. (d) Cluster of proliferated CD3+ T-cells (brown) after 10-day culturing of PBMCs, PC3 tumor cells, and BsAb. Tumor cells vanish within the first 3 days. (e) CD8+ T-cells (brown) in different frequency of occurrence in clusters surrounding DU145 cells (black). PBMCs surrounding the tumor cells are not dominated by one subpopulation (CD4+, CD8+, CD14+). Bars = 50 µm. (f) PBMCs (blue) partly expressing pore-forming perforin proteins (brown) concentrated towards the target cell (LNCaP, black). Bar = 10 µm.

CD3, CD4, CD8, and CD14 Immunocytochemistry
To investigate the activated T-cell subpopulations within the clusters of lymphocytes and the alterations of tumor cells, we applied immunocytochemical double staining. Chamber slide cultures were incubated up to 10 days without changing the medium conditions. Cells within the clusters predominantly proved to be CD3+ T-cells throughout the experimental period (Fig 1d). T-cells are characterized by a strong proliferating capacity increasing from Day 1 to Day 7, as indicated by Ki-67+ immunocytochemistry (data not shown). After a total time of 10 days, several huge accumulations of CD3+ T-cells had developed. At that time, tumor cells could no longer be observed. At Day 3, staining of cytokeratin as an indicator for epithelial cells was negative. In contrast, PBMCs and tumor cells incubated without bispecific antibodies obviously were growing independently of each other (Fig 1c). Formation of clusters and increasing proliferation were absent.

The frequency of occurrence of CD4+, CD8+, and CD14+ cells in the experimental sets was in accordance with the distribution in peripheral blood (CD4 > CD8 > CD14). Presumably the distribution is subjected to strong displacement after the necessary removal of culture medium from the wells, which inevitably leads to a loss of substantial quantity of PBMCs in the preparations. Within the first 3 days, i.e., the tumor elimination interval, PBMCs surrounding the tumor cells were not dominated by one of the three subpopulations mentioned above. We observed e.g., CD8+ cell clusters in addition to CD8+-free clusters (Fig 1e).

Perforin Immunocytochemistry
Although the culture medium has to be removed from the chamber slides before preparation of the slides for immunocytochemical investigations and therefore a possible destruction of cell formations cannot be avoided, lymphocytes can be found containing pore-forming perforin proteins, concentrated towards the tumor target cell (Fig 1f). Perforin is the presumed agent that perforates the cell membranes and provokes cell lysis, implying that perforin may play a role in lysis within the limits of our experiments.

Apoptosis
There was no evidence of apoptosis using three different methods. Neither antibodies for the detection of a caspase cleavage product of cytokeratin 18 (M30; Roche) or active caspase-3 (Pharmingen) nor the application of the classical TUNEL method (ApopTag; Intergen) resulted in identification of definitely apoptotic cells.

Computerized Sequential Video Microscopy
In response to the contact between effector and target cells induced by the trifunctinal bsAb, the adherent growing tumor cells shrank to approximately 50% of the original size, followed by a distinct swelling for 6–8 hr until the cell membrane was suddenly disrupted. Obviously, the increasing osmotic pressure was responsible for tumor cell lysis. This short-time event could be visualized in a quick-time video clip produced of the sequence of stored video frames. The entire process of tumor cell killing took at least 20 hr but no longer than 72 hr from the beginning of the experiment. Simultaneously, the PBMC proliferation and differentiation increased (Fig 2).



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Figure 2. Computerized sequential storage of video frames at 30-sec intervals reveals details of cell lysis. Adherent growing PC3 tumor cell (a, arrow) shrinks (b), subsequently swells (c), and finally disappears (d) within a minute. Another three tumor cells within this sequence undergo the same process temporarily delayed (e,h, arrows). Bar = 50 µm.


  Discussion
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Materials and Methods
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Literature Cited

Bispecific monoclonal antibodies of the trifunctional type bind specifically to a tumor-associated antigen (e.g., EpCAM) and the CD3 antigen on T-lymphocytes. In addition, the Fc region of such trifunctional antibodies binds and activates Fc{gamma}R+ accessory cells (e.g., monocytes, macrophages, dendritic cells; Zeidler et al. 1999 ), delivering the co-stimulatory signals which are needed by the T-cell for physiological activation according to the two-signal hypothesis (Schwartz 1989 ). In this context, it is noteworthy that targeting of antigens to specific Fc receptor molecules on antigen-presenting cells can markedly reduce the concentration of antigen required for a significant immunological response (Guyre et al. 1997 ). Obviously, the advantage of this class of bispecific antibodies over the conventional therapeutic antibodies is the fact that they activate two immunological cascades simultaneously and therefore are particularly effective in the destruction of tumor cells (Zeidler et al. 1999 ). However, we must stress that the role of the Fc portion in the anti-tumor processes presented here is at present only suggested due to the results of former experiments performed with other tumor cell lines (Zeidler et al. 1999 , Zeidler et al. 2000 ).

For the first time we investigated the {alpha}EpCAM x {alpha}CD3 trifunctional bsAb in a co-culture of EpCAM+ PC cells and PBMCs to determine if lymphocytes kill target cells by the perforin pathway or by an antibody-induced apoptotic pathway. Several authors reported heterogeneous apoptotic responses of LNCaP, PC3, and DU145 cells after treatment with camptothecin, staurosporine, phenlybutyrate, and two polyamine analogues (Wang et al. 1999 ; Marcelli et al. 2000 ; McCloskey et al. 2000 ; Ng et al. 2000 ). On the other hand, these cell lines express Fas (CD95) on the cell surface but are resistant to killing by anti-Fas antibody (Frost et al. 1997 ). Thus, the absence of any form of apoptotic cell death in all three cell lines in our investigations using three markers for apoptosis (TUNEL, M30 Cyto Death, anti-active caspase 3) may depend on a resistance to Fas-mediated apoptosis. This point will be of interest in further investigations using EpCAM+ cells susceptible to apoptosis by the Fas pathway. In this study, we observed osmotic lysis as a necrotic mode of cell death, which could be demonstrated by sequential video frame capture. Interestingly, the EpCAM x CD3 bsAb did not preferentially cluster the CD8+ or CD4+ subset. This observation is supported by previous reports that demonstrated proliferation and cytotoxic activity in both T-cell subpopulations for CD3 x EpCAM bsAb-targeted gastric cancer cells (Kufer et al. 1997 ) and for CD3 x CD19 bsAb, using B-cell lines as target cells in the cytotoxicity assays (Haagen et al. 1994 ).

The activity of effector lymphocytes after attaching at the target cells represented by perforin monomers (Fig 1f) causes osmotic cytolysis and the typical picture of necrosis (Griffiths 1995 ). Secreted granzymes enter the target cell via the perforin channel and initiate an internal disintegration pathway that leads to DNA fragmentation (apoptosis) (Nakajima and Henkart 1994 ). Obviously, an interruption of the relationship between perforin and granzymes and thus an interruption of the apoptotic cascade leads to a non-apoptotic method of cell death.

EM-visible pores may perturb the permeability of the target membrane and lead to osmotic lysis of target cells (Liu et al. 1995 ). It was recently demonstrated that granzyme B is secreted from cytotoxic cells as a macromolecular complex with the carrier proteoglycan, serglycin, and targets pulsed with the complexes undergo apoptosis after the addition of perforin (Browne et al. 1999 ). Only when extremely high concentrations of perforin (>4000 U/ml) were present was it possible to identify functional pores that were large enough to account for transmembrane entry of granzyme B into the cell to continue the apoptotic pathway (Browne et al. 1999 ).

In summary, the temporally as well as morphologically reproducible cell lysis is never related to apoptosis, whereas data support the view that osmotic lysis could be responsible for necrosis in our experiments. The first attempt to attack low immunogenic prostate carcinoma cells with T-lymphocytes and accessory cells, activated by trifunctional bsAb in vitro, is a reproducible approach on the way to successful systemic elimination of disseminated tumor cells.


  Acknowledgments

Supported in part by grant Ho 1596/3-1 from the Deutsche Forschungsgemeinschaft (DFG).

We thank Roswitha Fischer, Barbara Ganzmann, and Birgit Stadlbauer for skillful technical assistance and Karl Rohrmann for generously supplying PBMCs.

Received for publication November 6, 2000; accepted February 7, 2001.


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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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