Chapter 29 Functional Analysis of CD59 Using Complement-Dependent Cytotoxicity Assay Martin Kolev Abstract CD59 overexpression has been shown to confer the resistance of tumors to complement lysis. Complement lysis is one of the two major killing mechanisms of therapeutic anticancer antibodies. This chapter provides a method that allows studying the extent of complement protection of tumors by CD59. Key words Complement-dependent cytotoxicity, Fab fragments , Normal human serum , Anti- disialoganglioside GD2 antibody, Cancer, Cell lines
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Introduction Complement-dependent cytotoxicity (CDC) is a mechanism of complement activation in the presence of complement-activating antibodies. It is one of the main mechanisms of action of anticancer monoclonal antibodies [1, 2]. Rituximab (humanized antiCD20 antibody used in the treatment of non-Hodgkin lymphoma) has been shown to have CDC as one of the main mechanisms of action [3]. CDC is routinely employed to investigate the resistance of cancer cells to complement lysis. Limiting factor for CDC is the expression of membrane-bound complement regulators (mCReg), namely, CD46, CD55 and CD59 [2, 4]. A schematic representation of steps involved in CDC is shown in Fig. 1. Briefly, the target cells were first incubated with the fluorescent viability dye calcein AM for 30 min at 37 °C in complete RPMI 1640 media. Following two washes in plain RPMI 1640 (without serum), cells were incubated for 30 min at 4 °C with complement-activating antibody anti-disialoganglioside GD2 (short GD2), and in some cases Fab fragments of polyclonal antiCD59 antibody were also added. GD2 antigen is expressed on the
Mihaela Gadjeva (ed.), The Complement System: Methods and Protocols, Methods in Molecular Biology, vol. 1100, DOI 10.1007/978-1-62703-724-2_29, © Springer Science+Business Media New York 2014
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Fig. 1 Schematic representation of the steps involved in complement-dependent cytotoxicity assay. The assay is based on loading the cells with viability dye such as calcein AM prior to the binding of antibody. Serum is then added as a complement source. At the end of the assay free calcein AM in the cell supernatant is detected
surface of many tumor cells such as melanoma, neuroblastoma, and small cell lung carcinoma. Unbound antibody is then removed by washing with plain RPMI 1640 media, and a source of complement is added for 1 h at 37 °C. Normal human serum (NHS) was used as a source of complement. Cells are then pelleted and supernatants analyzed for fluorescence using plate reader. For alternative target cells refer to Note 1.
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Materials Please carry out all steps in sterile conditions under laminar flow cabinet unless otherwise stated to avoid contamination. Follow all waste disposal regulations when disposing waste materials. Typically no sodium azide was added to the reagents to ensure that cells are not harmed by its toxic effect. 1. RPMI 1640. Supplement the media with 10 % fetal calf serum, sodium pyruvate (5 mM), streptomycin (50 U/ml), penicillin (50 U/ml), and L-glutamine (2 mM). Store at 4 °C. 2. DMEM. Supplement the media as in item 1 for RPMI 1640. Store at 4 °C. 3. Tumor cell lines or primary cells to be used as target cells for complement-dependent lysis. We have used melanoma cell line G361 and neuroblastoma cell line IMR32. 4. Calcein AM. See Note recommendations.
1 for dilution and storage
5. NHS (see Note 2 for instructions on how to prepare NHS). Store at −80 °C. 6. Cell detaching buffer. 10 mM EDTA in PBS. Store at 4 °C.
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7. Cell lysis buffer. 10× solution, 2 % Tritone X100 in PBS ( see Note 2 for preparation instructions). Store at 4 °C. 8. Anti-disialoganglioside GD2 antibody (Millipore). Store at 4 °C. 9. Fab fragments of anti-CD59 antibody in-house made. Alternatively use any polyclonal anti-human CD59 to make sure that all epitopes of CD59 are blocked. See Note 3 for the protocol used for preparation of Fab fragments. Store at 4 °C. 10. 96-Well U- or V-bottom plates. 11. Plate reader capable of recoding fluorescence (we used FLUOstar Optima). 12. Incubator capable of maintaining 37 °C and 5 % CO2 humidified atmosphere.
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Methods Before starting the method follow instructions in Note 4 to prepare working calcein AM solution. Follow Notes 3 and 5 to prepare NHS and Fab fragments, respectively. Prepare a scheme of the experimental setup. Use Fig. 2 for guidance. 1. Grow the cells in appropriate media until 70 % confluent. IMR32 was maintained in RPMI 1640, while DMEM was used for G361 cells (for more information on cell lines see Note 6). DO NOT allow the cells to overgrow. 2. On the day before experiment replace the media with a fresh one.
Fig. 2 Example plate setup. The drawing represents a 96-well plate containing the experimental and control wells necessary for the CDC assay. Perform each experimental and control reaction in at least triplicate
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3. Next day add to the cells 5 ml cell detaching solution. Incubate for 5–10 min at 37 °C. 4. Wash the cells once by adding 10 ml 1× PBS to the cells from one T75 mm3 flask containing detaching solution. 5. Spin down the cells at 250 × g for 5 min at room temperature. 6. Remove the supernatant and resuspend in 10 ml 1× PBS. 7. Count the cells using hemocytometer or other suitable method. 8. Adjust the cell counts to 1 × 106 cells/ml in plain RPMI 1640. 9. Add 15.2 μl of stock calcein AM solution to 1 ml of the cellular suspension. For preparation of stock calcein AM see Note 4. 10. Incubate for 30 min at 37 °C. Resuspend the cells by gently tapping the vial every 10 min to ensure that the dye is loaded evenly. 11. Add plain RPMI 1640 up to 20 ml and spin down at 250 × g for 5 min at room temperature. Remove the supernatant. 12. Repeat step 11 once more. 13. Add 5 ml of plain RPMI 1640 to cell pellet and mix by pipetting up and down gently. 14. Dispense 50 μl of cells per well in 96-well U-bottom plate. When preparing for the assay, make sure to have spare wells for the controls as suggested in Fig. 2. Required controls are cells only in media and cells treated with cell lysis buffer (see Note 2, for preparation of stock solution, dilute ten times to working concentration (0.2 % Trition X100)). Cells with media are the background control for spontaneous release of the fluorescent dye in the media, while cells with 1× lysis buffer are the positive control for the maximum amount of fluorescence taken by the cells (for more information and how to calculate % of lysis see Note 7). We recommend preparing triplicates for each condition and control. 15. Prepare anti-GD2 and anti-CD59 Fab solutions. We used 5 μg/ml final concentration of anti-GD2 antibody and 100 μg/ml final concentration of anti-CD59 Fab (to ensure that all CD59 expressed on the surface is blocked). We recommend tittering the anti-CD59 antibody for each cell line used. 16. Dispense 50 μl of each anti-GD2 antibody and anti-CD59 Fab per well to bring the total volume to 100 μl. 17. Incubate for 30 min at 4 °C. 18. Wash once by adding 100 μl plain media and centrifuging for 5 min at 250 × g. Add 100 μl plain media to the wells.
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19. 5 min before the end of incubation time thaw the NHS in water bath and dilute it as required. Start from neat NHS (100 %), and make several twofold dilutions with RPMI 1640 to obtain dilution curve (see also Fig. 2 for recommended final dilutions of the serum). Alternatively the serum can be kept in the same concentration, and the GD2 antibody or the CD59 Fab fragments can be tittered. 20. Add 50 μl of undiluted or diluted NHS to the wells. Total volume then should be 150 μl. Also at this step add 35 μl of media and 15 μl of cell lysis buffer (to make 1×) to maximum release wells. For the background control wells add 50 μl plain media. 21. Spin down the plate for 5 min at 250 × g at room temperature. 22. Incubate for 1 h at 37 °C. 23. Spin down the plate at 250 × g for 5 min. 24. Using multichannel pipette remove very carefully 75 μl of the supernatant, and dispense it into fresh microtiter flat-bottom plate. DO NOT disturb the pellet. 25. Record fluorescence at 480/520 nm. Some typical cytotoxicity curves are given in Fig. 3. Refer to Note 7 for how to calculate the percent of lysis.
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Notes 1. Mouse or rat cells can be used in this assay. In these cases we recommend using mouse or rat serum as the source of complement. The other two complement regulators can also be blocked in order to dissect the mechanism of protection against complement in the same way as shown below. 2. Dilute 2 ml of 100 % stock Triton X100 in 98 ml PBS to obtain 2 % (10× concentrated) stock. Store at 4 °C. 3. Before starting the digestion, check the integrity of the antibody on SDS-PAGE. One mg of anti-CD59 (rabbit antihuman polyclonal antibody), made in-house against whole soluble CD59 molecule, is dialyzed against Fab digestion buffer (17 mM cysteine-HCl, 0.1 M Na2HPO4, 10 mM EDTA; pH 7.0) overnight at 4 °C and then incubated with 250 μl of a papain slurry (Pierce) for 2–3 h at 37 °C with shaking. Following incubation, the samples are centrifuged to remove the slurry. Digestion can then be verified on nonreducing and reducing 10 % SDS-PAGE gels, with Fab fragments appearing as 50 kDa band (nonreducing) or around 30 kDa (reducing conditions).
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Fig. 3 Typical killing curves. (a) G361 cells were incubated with GD2 antibody as per protocol. Serum was titrated from neat to 4.325 % using twofold dilutions. Percent of specific lysis was calculated as mean ± SD from each triplicate condition as explained in Note 6. (b) Same as for (a) but using a different target cell line—IMR32. (c) CDC assay with blocking Fabs. The assay was repeated in the same serum concentration (20 %), and cells were preincubated with either no Fabs or blocking Fab fragments against either CD59 or all mCregs (CD46, CD55, and CD59). Results indicate that the addition of either CD59 or all Fabs increased % of lysis significantly compared to non-treated cells indicating that these regulators protect the cells from complement killing
4. Dilute one 50 mg vial of calcein AM (Special packaging, Molecular Probes) with 50 μl sterile DMSO (1 mg/ml concentration) in sterile conditions. Use 15.6 μl of stock for 1 ml or 7.4 μl for 500 μl media. 5. NHS is prepared from fresh blood. Serum is blood plasma without fibrinogen or the other clotting factors. Collect blood
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in glass flasks, and allow clotting at room temperature for 1 h with occasional stirring using a glass stick. After clotting incubate the blood on ice for a further 30 min–1 h to promote clot contraction. After a 5-min spin at 650 × g in a bench-top centrifuge collect the serum supernatant and transfer into new vial for another 650 × g spin but this time for 10 min. Serum appearance should be yellow with little evidence of hemolysis. Aliquot the serum and store at −80 °C until required. Thaw only once to ensure maximal activity. 6. The cell lines listed are used due to their variable mCRegs and availability of complement fixing antibody—GD2 (supplied by Millipore). IMR32 only expresses very little CD46 and CD59, and while G361 expresses all mCReg, CD59 seems to be conferring its resistance to CDC. Other cell lines or primary cells can be adapted to be used with this protocol. 7. To calculate the percent of specific lysis use the following formula: % lysis = 100 × (TestF480/520 − 0 %F480/520)/(100 %F480/520 − 0 %F480/520) where Test—Fluorescence of the experimental wells 0 %—Fluorescence background in wells where only cells and media were present (background control) 100 %—Fluorescence of cells incubated with detergent to determine maximum killing (maximum release) References 1. Gelderman K, Tomlinson S, Ross G et al (2004) Complement function in mAb-mediated cancer immunotherapy. Trends Immunol 25:158–164 2. Kolev M, Towner L, Donev R (2011) Complement in cancer and cancer immunotherapy. Arch Immunol Ther Exp (Warsz) 59:407–419 3. Di Gaetano N, Cittera E, Nota R et al (2003) Complement activation determines the thera-
peutic activity of rituximab in vivo. J Immunol 171:1581–1587 4. Gelderman KA, Blok VT, Fleuren GJ et al (2002) The inhibitory effect of CD46, CD55, and CD59 on complement activation after immunotherapeutic treatment of cervical carcinoma cells with monoclonal antibodies or bispecific monoclonal antibodies. Lab Invest 82:483–493
1
Introduction Complement-dependent cytotoxicity (CDC) is a mechanism of complement activation in the presence of complement-activating antibodies. It is one of the main mechanisms of action of anticancer monoclonal antibodies [1, 2]. Rituximab (humanized antiCD20 antibody used in the treatment of non-Hodgkin lymphoma) has been shown to have CDC as one of the main mechanisms of action [3]. CDC is routinely employed to investigate the resistance of cancer cells to complement lysis. Limiting factor for CDC is the expression of membrane-bound complement regulators (mCReg), namely, CD46, CD55 and CD59 [2, 4]. A schematic representation of steps involved in CDC is shown in Fig. 1. Briefly, the target cells were first incubated with the fluorescent viability dye calcein AM for 30 min at 37 °C in complete RPMI 1640 media. Following two washes in plain RPMI 1640 (without serum), cells were incubated for 30 min at 4 °C with complement-activating antibody anti-disialoganglioside GD2 (short GD2), and in some cases Fab fragments of polyclonal antiCD59 antibody were also added. GD2 antigen is expressed on the
Mihaela Gadjeva (ed.), The Complement System: Methods and Protocols, Methods in Molecular Biology, vol. 1100, DOI 10.1007/978-1-62703-724-2_29, © Springer Science+Business Media New York 2014
347
348
Martin Kolev
Fig. 1 Schematic representation of the steps involved in complement-dependent cytotoxicity assay. The assay is based on loading the cells with viability dye such as calcein AM prior to the binding of antibody. Serum is then added as a complement source. At the end of the assay free calcein AM in the cell supernatant is detected
surface of many tumor cells such as melanoma, neuroblastoma, and small cell lung carcinoma. Unbound antibody is then removed by washing with plain RPMI 1640 media, and a source of complement is added for 1 h at 37 °C. Normal human serum (NHS) was used as a source of complement. Cells are then pelleted and supernatants analyzed for fluorescence using plate reader. For alternative target cells refer to Note 1.
2
Materials Please carry out all steps in sterile conditions under laminar flow cabinet unless otherwise stated to avoid contamination. Follow all waste disposal regulations when disposing waste materials. Typically no sodium azide was added to the reagents to ensure that cells are not harmed by its toxic effect. 1. RPMI 1640. Supplement the media with 10 % fetal calf serum, sodium pyruvate (5 mM), streptomycin (50 U/ml), penicillin (50 U/ml), and L-glutamine (2 mM). Store at 4 °C. 2. DMEM. Supplement the media as in item 1 for RPMI 1640. Store at 4 °C. 3. Tumor cell lines or primary cells to be used as target cells for complement-dependent lysis. We have used melanoma cell line G361 and neuroblastoma cell line IMR32. 4. Calcein AM. See Note recommendations.
1 for dilution and storage
5. NHS (see Note 2 for instructions on how to prepare NHS). Store at −80 °C. 6. Cell detaching buffer. 10 mM EDTA in PBS. Store at 4 °C.
Assay for Measuring Lysis of Cancer Cells by Complement
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7. Cell lysis buffer. 10× solution, 2 % Tritone X100 in PBS ( see Note 2 for preparation instructions). Store at 4 °C. 8. Anti-disialoganglioside GD2 antibody (Millipore). Store at 4 °C. 9. Fab fragments of anti-CD59 antibody in-house made. Alternatively use any polyclonal anti-human CD59 to make sure that all epitopes of CD59 are blocked. See Note 3 for the protocol used for preparation of Fab fragments. Store at 4 °C. 10. 96-Well U- or V-bottom plates. 11. Plate reader capable of recoding fluorescence (we used FLUOstar Optima). 12. Incubator capable of maintaining 37 °C and 5 % CO2 humidified atmosphere.
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Methods Before starting the method follow instructions in Note 4 to prepare working calcein AM solution. Follow Notes 3 and 5 to prepare NHS and Fab fragments, respectively. Prepare a scheme of the experimental setup. Use Fig. 2 for guidance. 1. Grow the cells in appropriate media until 70 % confluent. IMR32 was maintained in RPMI 1640, while DMEM was used for G361 cells (for more information on cell lines see Note 6). DO NOT allow the cells to overgrow. 2. On the day before experiment replace the media with a fresh one.
Fig. 2 Example plate setup. The drawing represents a 96-well plate containing the experimental and control wells necessary for the CDC assay. Perform each experimental and control reaction in at least triplicate
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3. Next day add to the cells 5 ml cell detaching solution. Incubate for 5–10 min at 37 °C. 4. Wash the cells once by adding 10 ml 1× PBS to the cells from one T75 mm3 flask containing detaching solution. 5. Spin down the cells at 250 × g for 5 min at room temperature. 6. Remove the supernatant and resuspend in 10 ml 1× PBS. 7. Count the cells using hemocytometer or other suitable method. 8. Adjust the cell counts to 1 × 106 cells/ml in plain RPMI 1640. 9. Add 15.2 μl of stock calcein AM solution to 1 ml of the cellular suspension. For preparation of stock calcein AM see Note 4. 10. Incubate for 30 min at 37 °C. Resuspend the cells by gently tapping the vial every 10 min to ensure that the dye is loaded evenly. 11. Add plain RPMI 1640 up to 20 ml and spin down at 250 × g for 5 min at room temperature. Remove the supernatant. 12. Repeat step 11 once more. 13. Add 5 ml of plain RPMI 1640 to cell pellet and mix by pipetting up and down gently. 14. Dispense 50 μl of cells per well in 96-well U-bottom plate. When preparing for the assay, make sure to have spare wells for the controls as suggested in Fig. 2. Required controls are cells only in media and cells treated with cell lysis buffer (see Note 2, for preparation of stock solution, dilute ten times to working concentration (0.2 % Trition X100)). Cells with media are the background control for spontaneous release of the fluorescent dye in the media, while cells with 1× lysis buffer are the positive control for the maximum amount of fluorescence taken by the cells (for more information and how to calculate % of lysis see Note 7). We recommend preparing triplicates for each condition and control. 15. Prepare anti-GD2 and anti-CD59 Fab solutions. We used 5 μg/ml final concentration of anti-GD2 antibody and 100 μg/ml final concentration of anti-CD59 Fab (to ensure that all CD59 expressed on the surface is blocked). We recommend tittering the anti-CD59 antibody for each cell line used. 16. Dispense 50 μl of each anti-GD2 antibody and anti-CD59 Fab per well to bring the total volume to 100 μl. 17. Incubate for 30 min at 4 °C. 18. Wash once by adding 100 μl plain media and centrifuging for 5 min at 250 × g. Add 100 μl plain media to the wells.
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19. 5 min before the end of incubation time thaw the NHS in water bath and dilute it as required. Start from neat NHS (100 %), and make several twofold dilutions with RPMI 1640 to obtain dilution curve (see also Fig. 2 for recommended final dilutions of the serum). Alternatively the serum can be kept in the same concentration, and the GD2 antibody or the CD59 Fab fragments can be tittered. 20. Add 50 μl of undiluted or diluted NHS to the wells. Total volume then should be 150 μl. Also at this step add 35 μl of media and 15 μl of cell lysis buffer (to make 1×) to maximum release wells. For the background control wells add 50 μl plain media. 21. Spin down the plate for 5 min at 250 × g at room temperature. 22. Incubate for 1 h at 37 °C. 23. Spin down the plate at 250 × g for 5 min. 24. Using multichannel pipette remove very carefully 75 μl of the supernatant, and dispense it into fresh microtiter flat-bottom plate. DO NOT disturb the pellet. 25. Record fluorescence at 480/520 nm. Some typical cytotoxicity curves are given in Fig. 3. Refer to Note 7 for how to calculate the percent of lysis.
4
Notes 1. Mouse or rat cells can be used in this assay. In these cases we recommend using mouse or rat serum as the source of complement. The other two complement regulators can also be blocked in order to dissect the mechanism of protection against complement in the same way as shown below. 2. Dilute 2 ml of 100 % stock Triton X100 in 98 ml PBS to obtain 2 % (10× concentrated) stock. Store at 4 °C. 3. Before starting the digestion, check the integrity of the antibody on SDS-PAGE. One mg of anti-CD59 (rabbit antihuman polyclonal antibody), made in-house against whole soluble CD59 molecule, is dialyzed against Fab digestion buffer (17 mM cysteine-HCl, 0.1 M Na2HPO4, 10 mM EDTA; pH 7.0) overnight at 4 °C and then incubated with 250 μl of a papain slurry (Pierce) for 2–3 h at 37 °C with shaking. Following incubation, the samples are centrifuged to remove the slurry. Digestion can then be verified on nonreducing and reducing 10 % SDS-PAGE gels, with Fab fragments appearing as 50 kDa band (nonreducing) or around 30 kDa (reducing conditions).
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Fig. 3 Typical killing curves. (a) G361 cells were incubated with GD2 antibody as per protocol. Serum was titrated from neat to 4.325 % using twofold dilutions. Percent of specific lysis was calculated as mean ± SD from each triplicate condition as explained in Note 6. (b) Same as for (a) but using a different target cell line—IMR32. (c) CDC assay with blocking Fabs. The assay was repeated in the same serum concentration (20 %), and cells were preincubated with either no Fabs or blocking Fab fragments against either CD59 or all mCregs (CD46, CD55, and CD59). Results indicate that the addition of either CD59 or all Fabs increased % of lysis significantly compared to non-treated cells indicating that these regulators protect the cells from complement killing
4. Dilute one 50 mg vial of calcein AM (Special packaging, Molecular Probes) with 50 μl sterile DMSO (1 mg/ml concentration) in sterile conditions. Use 15.6 μl of stock for 1 ml or 7.4 μl for 500 μl media. 5. NHS is prepared from fresh blood. Serum is blood plasma without fibrinogen or the other clotting factors. Collect blood
Assay for Measuring Lysis of Cancer Cells by Complement
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in glass flasks, and allow clotting at room temperature for 1 h with occasional stirring using a glass stick. After clotting incubate the blood on ice for a further 30 min–1 h to promote clot contraction. After a 5-min spin at 650 × g in a bench-top centrifuge collect the serum supernatant and transfer into new vial for another 650 × g spin but this time for 10 min. Serum appearance should be yellow with little evidence of hemolysis. Aliquot the serum and store at −80 °C until required. Thaw only once to ensure maximal activity. 6. The cell lines listed are used due to their variable mCRegs and availability of complement fixing antibody—GD2 (supplied by Millipore). IMR32 only expresses very little CD46 and CD59, and while G361 expresses all mCReg, CD59 seems to be conferring its resistance to CDC. Other cell lines or primary cells can be adapted to be used with this protocol. 7. To calculate the percent of specific lysis use the following formula: % lysis = 100 × (TestF480/520 − 0 %F480/520)/(100 %F480/520 − 0 %F480/520) where Test—Fluorescence of the experimental wells 0 %—Fluorescence background in wells where only cells and media were present (background control) 100 %—Fluorescence of cells incubated with detergent to determine maximum killing (maximum release) References 1. Gelderman K, Tomlinson S, Ross G et al (2004) Complement function in mAb-mediated cancer immunotherapy. Trends Immunol 25:158–164 2. Kolev M, Towner L, Donev R (2011) Complement in cancer and cancer immunotherapy. Arch Immunol Ther Exp (Warsz) 59:407–419 3. Di Gaetano N, Cittera E, Nota R et al (2003) Complement activation determines the thera-
peutic activity of rituximab in vivo. J Immunol 171:1581–1587 4. Gelderman KA, Blok VT, Fleuren GJ et al (2002) The inhibitory effect of CD46, CD55, and CD59 on complement activation after immunotherapeutic treatment of cervical carcinoma cells with monoclonal antibodies or bispecific monoclonal antibodies. Lab Invest 82:483–493
