LV received funding from National Research, Development and Innovation Office grants GINOP-2.3.2-15-2016-00010 TUMORDNS&#34;, GINOP-2.3.2-15-2016-00048-STAYALIVE and OTKA K132193, K147482. CD16.176V.NK-92 cells were obtained from Dr. Kerry S. Campbell (Fox Chase Center, Philapedlphia, PA, on behalf of Brink Biologics, lnc. San Diego, CA), are protected by patents worldwide, and were licensed by Nantkwest, lnc. Authors are thankful to Gy&#246;rgy Vereb and &#193;rp&#225;d Sz&#246;&#337;r for their help with the use of the NK-92 cell line and for technical advice.

NOTE: Key steps of the assay workflow are presented in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64485/64485fig02.jpg" /> 
Figure 2: Workflow of the ADCC screen. JIMT-1-EGFP target cells seeded into 96 well HCS plates are treated with drugs of the compound library. In turn, unstained NK (effector) cells and trastuzumab are added, and the plate is imaged at 0 timepoint and after 3 h of incubation. ADCC evaluation is based on the change in the number of viable (surface adherent) target cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64485/64485fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Coating of the HCS plate
<ol>
	<li>Coat the 96 well high-content screening (HSC) plates with 50 &#181;L/well JIMT-1 medium (DMEM/F-12 medium supplemented with 20% fetal bovine serum (FBS), 0.3 U/mL insulin (100 IU/mL, Humulin R, and 1% penicillin-streptomycin).</li>
	<li>Place the plate into a CO2 incubator for 1 h. 
	&#8203;NOTE: Coating is crucial for attaching JIMT-1 cells to the glass surface of the plate.</li>
</ol>
2. Seeding of JIMT-1 Enhanced Green Fluorescent Protein (EGFP) cells
NOTE: EGFP-expressing JIMT-1 cells were generated in our previous work18, and the cells were cultured in T25 tissue culture flasks in JIMT-1 media (see composition in step 1.1).
<ol>
	<li>Wash the cells with 2 mL of sterile PBS.</li>
	<li>Add 1 mL of trypsin-EDTA to the flask and put the flask back into a CO2 incubator for 10 min.</li>
	<li>After the incubation, tap the flask to check if JIMT-1 cells are detached.</li>
	<li>Stop the digestion with 2 mL of JIMT-1 media and collect the cell suspension into a 15 mL tube.</li>
	<li>Count the cells with 0.4% trypan blue (80 &#181;L of the dye + 20 &#181;L of the cell suspension) in a B&#252;rker chamber and adjust the cell number to 133, 000 cells/mL.</li>
	<li>Aspirate the coating medium from the 96 well plate (step 1.2).</li>
	<li>Pipette 75 &#181;L of the cell suspension to each well of the HCS plates (see Table of Materials).</li>
	<li>Allow cells to attach during an overnight incubation at 37 &#176;C in a CO2 incubator.</li>
</ol>
3. Pre-treatment of JIMT-1 EGFP cells with the compound library
<ol>
	<li>Aspirate the medium from the JIMT-1 cells and add 50 &#181;L/well fresh JIMT-1 medium to the wells. Transfer the plate to the high-throughput screening laboratory. 
	NOTE: Using a liquid handling robot makes the addition of compound libraries more efficient and reproducible.</li>
	<li>Transfer the test compounds from the compound library plate to the assay plate with a pin tool calibrated to a 25 nL volume. Perform this four times. Four rounds of transfer give a final volume of 100 nL (and a 20 &#181;M final concentration).</li>
	<li>Between each step, wash the pin tool, first with 50% DMSO and then with 70% ethanol.</li>
	<li>Incubate the plates for 1 h in a CO2 incubator at 37 &#176;C.</li>
</ol>
4. Starting the ADCC assay by adding the effector cells
NOTE: CD16.176V.NK92 cells (hereafter referred to as NK92 cells)&#160;were cultured in &#945;-MEM supplemented with 20% FBS, 1% MEM-NEAA, 1% Na-pyruvate, 1% glutamine, 1% penicillin-streptomycin and 100 IU/mL IL-2.
<ol>
	<li>Count NK92 cells with trypan blue (80 &#181;L of the dye + 20 &#181;L of the cell suspension). Adjust the cell number to 400,000 cells/mL.</li>
	<li>Centrifuge 4 mL of the cell suspension at 150 x g for 3 min at room temperature.</li>
	<li>Prepare the ADCC medium by adding 20 &#181;g/mL anti-HER2 antibody (trastuzumab) to the JIMT-1 medium.</li>
	<li>Resuspend the NK cell pellet in 5 mL ADCC medium.</li>
	<li>Pipette 20,000 NK cells in 50 &#181;L of ADCC medium to the target JIMT-1 cells. The final volume is 100 &#181;L, and the final trastuzumab concentration is 10 &#181;g/mL.</li>
	<li>Place the assay plate into the high-content analysis equipment with a built-in incubator set at 37 &#176;C.</li>
</ol>
5. Imaging
NOTE: The plates should be imaged at two time points, first, immediately after the addition of the effector cells to the target cells and second, at 3 h after the addition of NK cells. For imaging, the high-content analyzer and its software or suitable alternatives can be used (see Table of materials).
<ol>
	<li>Select Plate type (96-well cell carrier ultra) from the list of plates.</li>
	<li>Select the Two peak autofocus if the assay is carried out in plates.</li>
	<li>Use 10x objective in non-confocal mode.</li>
	<li>Select Binning 2 to double the signal to noise ratio.</li>
	<li>Take brightfield images at 650-760 nm and fluorescent images of the EGFP-transduced JIMT-1 cells at 488 nm (excitation) and 500-550 nm (emission) wavelengths.</li>
	<li>Select the number of fields and the number of timepoints for the imaging.</li>
</ol>
6. Image analysis
NOTE: To analyze the ADCC efficiency, the viable JIMT-1 cells are counted. Target cells killed by ADCC detach from the surface and move away from the focal plane of the microscope. Therefore, the difference between the number of viable cells at the beginning and at the end of the ADCC reaction corresponds to target cells eliminated by ADCC. To show how to build up the evaluation sequence, a control ADCC well is shown in the video.
<ol>
	<li>Use the Find cells module to detect regions on the image that correspond to cells. 
	NOTE: Each cell is detected as a region on the image with a higher fluorescence intensity than its surrounding.</li>
	<li>Select cells using the built-in M algorithm with a minimum of 80 &#181;m in diameter.</li>
	<li>Set Splitting sensitivity, which parcels out a large object into smaller objects, to 0.5.</li>
	<li>Set the Common threshold (the lowest level of pixel intensity) to 0.</li>
	<li>Exclude the detection of background area with high EGFP fluorescence intensity in two steps.
	<ol>
		<li>First, use the Calculate Intensity Properties function to determine the EGFP fluorescence intensity in the previously selected Cells region.</li>
		<li>Set the minimum and maximum intensity threshold using the Select population option.</li>
	</ol>
	</li>
</ol>

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<li>Barok, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trastuzumab+causes+antibody-dependent+cellular+cytotoxicity-mediated+growth+inhibition+of+submacroscopic+JIMT-1+breast+cancer+xenografts+despite+intrinsic+drug+resistance%5BTitle%5D)+AND+%22Molecular+and+Cancer+Therapy%22%5BJournal%5D)">Trastuzumab causes antibody-dependent cellular cytotoxicity-mediated growth inhibition of submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug resistance.</a> Molecular and Cancer Therapy. 6 (7), 2065-2072 (2007).</li>
<li>Gauthier, M., Laroye, C., Bensoussan, D., Boura, C., Decot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Natural+Killer+cells+and+monoclonal+antibodies%3A+Two+partners+for+successful+antibody+dependent+cytotoxicity+against+tumor+cells%5BTitle%5D)+AND+%22Crit+Rev+Oncol+Hematol%22%5BJournal%5D)">Natural Killer cells and monoclonal antibodies: Two partners for successful antibody dependent cytotoxicity against tumor cells.</a> Crit Rev Oncol Hematol. 160, 103261(2021).</li>
<li>Gruijs, M., Sewnath, C. A. N., van Egmond, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+exploitation+of+neutrophils+to+fight+cancer%5BTitle%5D)+AND+%22Semin+Immunol%22%5BJournal%5D)">Therapeutic exploitation of neutrophils to fight cancer.</a> Semin Immunol. 57, 101581(2021).</li>
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<li>Broussas, M., Broyer, L., Goetsch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+antibody-dependent+cell+cytotoxicity+using+lactate+dehydrogenase+%28LDH%29+measurement%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Evaluation of antibody-dependent cell cytotoxicity using lactate dehydrogenase (LDH) measurement.</a> Methods in Molecular Biology. 988, 305-317 (2013).</li>
<li>Toth, G., Szollosi, J., Vereb, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitating+ADCC+against+adherent+cells%3A+Impedance-based+detection+is+superior+to+release%2C+membrane+permeability%2C+or+caspase+activation+assays+in+resolving+antibody+dose+response%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Quantitating ADCC against adherent cells: Impedance-based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response.</a> Cytometry A. 91 (10), 1021-1029 (2017).</li>
<li>Chung, S., Nguyen, V., Lin, Y. L., Kamen, L., Song, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thaw-and-use+target+cells+pre-labeled+with+calcein+AM+for+antibody-dependent+cell-mediated+cytotoxicity+assays%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">Thaw-and-use target cells pre-labeled with calcein AM for antibody-dependent cell-mediated cytotoxicity assays.</a> Journal of Immunological Methods. 447, 37-46 (2017).</li>
<li>Lee-MacAry, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+novel+flow+cytometric+cell-mediated+cytotoxicity+assay+using+the+fluorophores+PKH-26+and+TO-PRO-3+iodide%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide.</a> Journal of Immunological Methods. 252 (1-2), 83-92 (2001).</li>
<li>Tanito, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+evaluation+of+natural+killer+cell-mediated+cell+killing+assay+based+on+the+leakage+of+an+endogenous+enzyme+or+a+pre-loaded+fluorophore%5BTitle%5D)+AND+%22Analytical+Science%22%5BJournal%5D)">Comparative evaluation of natural killer cell-mediated cell killing assay based on the leakage of an endogenous enzyme or a pre-loaded fluorophore.</a> Analytical Science. 37 (11), 1571-1575 (2021).</li>
<li>Lin, S., Schorpp, K., Rothenaigner, I., Hadian, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image-based+high-content+screening+in+drug+discovery%5BTitle%5D)+AND+%22Drug+Discovery+Today%22%5BJournal%5D)">Image-based high-content screening in drug discovery.</a> Drug Discovery Today. 25 (8), 1348-1361 (2020).</li>
<li>Guti, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+multitargeted+receptor+tyrosine+kinase+inhibitor+sunitinib+induces+resistance+of+HER2+positive+breast+cancer+cells+to+trastuzumab-mediated+ADCC%5BTitle%5D)+AND+%22Cancer+Immunology%2C+Immunotherapy%22%5BJournal%5D)">The multitargeted receptor tyrosine kinase inhibitor sunitinib induces resistance of HER2 positive breast cancer cells to trastuzumab-mediated ADCC.</a> Cancer Immunology, Immunotherapy. 71 (9), 2151-2168 (2022).</li>
<li>Li, F., Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Focusing+on+NK+cells+and+ADCC%3A+A+promising+immunotherapy+approach+in+targeted+therapy+for+HER2-positive+breast+cancer%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Focusing on NK cells and ADCC: A promising immunotherapy approach in targeted therapy for HER2-positive breast cancer.</a> Frontiers in Immunology. 13, 1083462(2022).</li>
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Authors report no conflict of interest.

The ADCC reaction has been described a relatively long time ago. Key molecular events of the process have also been described19. Methods for measuring ADCC range from the gold standard radioactive chromium release assay, cytoplasmic enzyme release assays to several fluorescence-based flow cytometry or microplate assays20. However, a common limitation of these assays is that they are not amenable to high-throughput applications. Previously, we developed an image-based HCS assay suitable for screening compound libraries for ADCC-modifying drugs18. The previously developed assay, however, had a major drawback i.e., it required a washing step because the target cells had to be pre-stained with calcein-acetoxymethylesther, and the excess dye had to be removed before the addition of the NK cells and trastuzumab. The current method represents an advanced version of the assay in that the use of EGFP-transduced JIMT-1 cells permits straightforward differentiation between effector and target cells without a staining and washing step. Moreover, one must also be aware of the feature of our assay that it cannot distinguish between dead and dying cells (not yet fully detached). Furthermore, it is possible that the test compounds may increase cell adhesion, and, as a result cell debris may remain in the focal plane resulting in a false positive result. In addition, the fluorescent properties of test compounds may cause interference. Therefore, although screens are typically run once, it is highly recommended to validate hits in other types of assays as well. Additional limitations of our assay include the indirect identification of cell death i.e., the method is based on the quantification of the change in the number of viable cells instead of dead cells. However, it is important to note here that a previous study found that ECIS-based detection of viable cells was superior to some of the methods directly measuring cell death13. Therefore, this feature of our method does not necessarily represent a drawback. What may really limit the application of this assay, however, is that it requires at least a basic knowledge in advanced image analysis, a skill that is becoming increasingly common today.
Although so far, a small library (less than 1000 compounds) has been tested with the original version of the assay18. We have not yet identified an ADCC booster drug. The reason for this might be that ADCC activators might be rare, and much bigger libraries need to be screened to identify one. Theoretically, it is also possible that once the ADCC reaction starts, it proceeds without major limiting steps so that the process would be difficult to improve further by a pharmacon. One possible way around this problem might be to suppress ADCC activity (e.g., with corticosteroids) and run the screen looking for compounds restoring full ADCC activity. Nevertheless, we think that identifying ADCC inhibitory effect of known medical drugs may also be important in order to raise awareness of clinicians prescribing these drugs to patients with various indications.
As for the technical details of the assay, we recommend setting up the assay in parallel with an alternative cytotoxicity test. In our hand, ECIS (electric cell-substrate impedance sensing) proved to be the most reliable system for the quantification of ADCC efficiency13. At the start of the project, ECIS was used to adjust critical parameters (time, effector to target cell ratio, trastuzumab concentration) and then transferred the assay to the HCS platform18. We recommend doing the same and fine tune these parameters, as they might change from lab to lab. When setting up the assay, it is important to pay special attention to some of the critical steps of the procedure. Standardizing culture conditions (splitting frequency, cell feeding, plating consistency) for both NK cells and target cells may increase reproducibility of ADCC efficiency. Moreover, suspending NK cell cultures must be performed with care as these cells tend to be sensitive to mechanical stress (Guti et al. unpublished observation). As for the compound library, it is important to make sure that all wells contain equal volumes of test compounds if pin tools are used for the transfer of compounds from the library plate to the assay plate. This may prevent varying amounts of compound solutions from sticking to the outer side of the pins. Furthermore, hit compounds identified in a screen should be validated preferably in a reliable assay based on a different principle. For this, we recommend the ECIS-based method11 (see above in the Introduction).
In conclusion, we set up a JIMT-1 EGFP-based in vitro ADCC assay system suitable for testing compound libraries with automated image analysis. The method was suitable for the identification of pharmacons with known ADCC modifying effects. The method is potentially suitable for the determination of cancer cell death caused by trastuzumab drug conjugates (e.g., recently developed trastuzumab-emtansine21) where the mechanism of toxicity is more complex and is only partially caused by ADCC and partially by the tubulin inhibitor mertansine. In the future, we plan to adopt the technology to quantify ADCC in 3D spheroids, which represent a more accurate reflection of tumor behavior than 2D models.

Immunotherapy with anticancer antibodies, immune checkpoint inhibitors, or chimeric antigen receptor-expressing T (CAR-T) cells represents a powerful approach to cancer treatment1,2,3. Trastuzumab is a humanized monoclonal anti-HER-2 (human epidermal growth factor receptor 2) antibody used for treating HER-2 positive early stage or metastatic breast cancer, as well as HER-2 positive metastatic gastric cancer4,5,6. It primarily acts by inhibiting the proliferation stimulating effect of the epidermal growth factor4. It has been reported, however, that trastuzumab efficiently triggers cancer cell death even if the cancer cells have lost their responsiveness to HER-2 stimulation7. This paradoxical effect of the antibody is due to antibody-dependent cell-mediated cytotoxicity (ADCC)7. ADCC can be mediated by natural killer (NK) cells, granulocytes, and macrophages collectively known as the effector cells of ADCC8,9. If an antibody, such as trastuzumab, binds to tumor cells, then these effector cells use their Fc receptors to bind the constant (Fc) region of the antibody. The antibody bridges the tumor cells and the Fc receptor-bearing effector cells, triggering the release of their cytotoxic mediators10. Natural killer cells release the cytotoxic cargo of their granules containing perforin to generate pores in the target cell membrane and granzyme (triggering cell death signaling pathways) into the immune synapse leading to apoptosis of the cancer cells (see Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64485/64485fig01.jpg" /> 
Figure 1: Effector and target cell interactions in ADCC. The cell surface Fc&#947; receptor of the effector NK cell recognizes the Fc region of the anti-HER2 trastuzumab antibody specific for the HER2 molecule expressed on the surface of the tumor cell. Thus, the so-called immunological synapse is established between the two cells, inducing the directed exocytosis of cytotoxic granules of the effector cell. The released perforin and granzyme molecules eventually result in apoptosis of the target cell. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64485/64485fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Several assays have previously been developed to quantify cytotoxicity, including ADCC. The gold standard is the radioactive chromium release method, where the target cells are labeled with radioactive 51Cr isotope, and ADCC is quantified by measuring radioactivity from the supernatant of lysed target cells11. Because of the obvious problems due to the strictly regulated handling, storage, and disposal of radioactive pharmacons and wastes, this method has become increasingly non-popular among life scientists. In addition, it is not amenable to high-throughput applications either. Measuring the activity of enzymes (e.g., lactate-dehydrogenase) released from the killed target cells can provide a non-radioactive alternative to the 51Cr assay12. These assays, however, fail to distinguish between target and effector cell deaths. Electric Cell-substrate Impedance Sensing (ECIS) proved suitable for the quantification of ADCC13, but the ECIS equipment is not available in most laboratories, and the technique is not compatible with high-throughput applications/screening. Fluorescently labeled cells represent a popular alternative in many cell biology assays and are often used in flow cytometry or plate reader-based applications14,15,16. However, these assays often contain washing steps or are otherwise incompatible with high-throughput applications (e.g., flow cytometry-based techniques). Some popular cytotoxicity assays, which in theory should be suitable for ADCC quantification, fail to reliably determine ADCC efficiency13. Recently, with the spreading of fluorescent confocal microscopy, image-based, high-content assays are becoming increasingly popular in various areas of life sciences17. On the one hand, cell imaging equipment are now rather ubiquitous, while, on the other hand, virtually endless morphological parameters can be gathered from the acquired images. Therefore, we set out to develop a high-content screening compatible ADCC assay and to demonstrate its suitability for compound library screening.
Here, we present an image-based ADCC assay and demonstrate how this assay can be used for High-Content Screening (HCS) to identify ADCC modulating compounds. The model is based on JIMT-1 breast carcinoma target cells, CD16.176V.NK-92 effector cells and the humanized monoclonal anti-HER2 antibody trastuzumab. With this method, it is possible to identify drugs that can enhance the tumor-killing action of NK cells or to gain insight into the mechanism of NK cell-mediated ADCC by identifying small molecules interfering with ADCC. We suggest that life scientists aiming to quantify cell-mediated cytotoxicity with special regard to ADCC may benefit from using this assay either for the discovery science or drug development. This assay may be an alternative if a laboratory has access to and some experience in fluorescent imaging and quantitative image analysis.

<table><tbody><tr><td>5-fluorouracil</td><td>Applichem</td><td>A7686</td><td>in compound library</td></tr><tr><td>96-well Cell Carrier Ultra plate</td><td>PerkinElmer</td><td>LLC 6055302</td><td></td></tr><tr><td>Betulin</td><td>Sigma</td><td>B9757</td><td>in compound library</td></tr><tr><td>CD16.176V.NK92 cells</td><td>Nankwest Inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td>Cerulenin</td><td>ChemCruz</td><td>sc-396822</td><td>in compound library</td></tr><tr><td>Cisplatin</td><td>Santa Cruz Biotechnology</td><td>sc-200896</td><td>in compound library</td></tr><tr><td>Colchicine</td><td>Sigma</td><td>C9754</td><td>in compound library</td></tr><tr><td>Concanavalin-A</td><td>Calbiochem</td><td>234567</td><td>in compound library</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td>in compound library</td></tr><tr><td>DMEM/F-12 medium</td><td>Sigma</td><td>D8437</td><td>in JIMT-1 EGFP medium</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td>in compound library</td></tr><tr><td>Etoposide</td><td>Sigma</td><td>E1383</td><td>E1383</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Biosera</td><td>FB-1090/500</td><td>JIMT-1 EGFP and NK medium</td></tr><tr><td>Fisetin</td><td>Sigma</td><td>F4043</td><td>in compound library</td></tr><tr><td>Freedom EVO liquid handling robot</td><td>TECAN</td><td></td><td></td></tr><tr><td>Gallotannin</td><td>Fluka Chemical Corp.</td><td>16201</td><td>in compound library</td></tr><tr><td>Glutamine</td><td>Gibco</td><td>35,050&amp;ndash;061</td><td>in NK medium</td></tr><tr><td>Harmony software&amp;nbsp;</td><td>PerkinElmer</td><td></td><td></td></tr><tr><td>Humanized anti-HER2 monoclonal antibody (Herzuma)</td><td>EGIS Pharmaceuticals, Budapest Hungary</td><td>N/A</td><td></td></tr><tr><td>Humulin R (insulin)</td><td>Eli Lilly</td><td>HI0219</td><td>JIMT-1 EGFP medium</td></tr><tr><td>IL-2</td><td>Novartis Hung&amp;aacute;ria Kft.</td><td>PHC0026</td><td>in NK medium</td></tr><tr><td>Isatin</td><td>Sigma</td><td>114618</td><td>in compound library</td></tr><tr><td>MEM Non-essential Amino Acids (MEM-NEAA)</td><td>Gibco</td><td>11,140&amp;ndash;050</td><td>in NK medium</td></tr><tr><td>Na-pyruvate</td><td>Lonza</td><td>BE13-115E</td><td>in NK medium</td></tr><tr><td>Naringenin</td><td>Sigma</td><td>N5893</td><td>in compound library</td></tr><tr><td>NQDI-1</td><td>Sigma</td><td>SML0185</td><td>in compound library</td></tr><tr><td>Opera Phenix High-Content Analysis equipment</td><td>PerkinElmer</td><td></td><td></td></tr><tr><td>Penicillin&amp;ndash;streptomycin</td><td>Biosera</td><td>LM-A4118</td><td>JIMT-1 EGFP and NK medium</td></tr><tr><td>Pentoxyfilline</td><td>Sigma</td><td>P1784</td><td>in compound library</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Lonza</td><td>BE17-517Q</td><td>to wash the cells</td></tr><tr><td>Podophyllotoxin</td><td>Sigma</td><td>P4405</td><td>in compound library</td></tr><tr><td>Quercetin</td><td>Sigma</td><td>Q4951</td><td>in compound library</td></tr><tr><td>Tannic acid</td><td>Sigma</td><td>T8406</td><td>in compound library</td></tr><tr><td>Temozolomide</td><td>Sigma</td><td>T2577</td><td>in compound library</td></tr><tr><td>Trypan blue 0.4% solution</td><td>Sigma</td><td>T8154</td><td>for cell counting</td></tr><tr><td>Vincristine sulfate</td><td>Sigma</td><td>V0400000</td><td>in compound library</td></tr><tr><td>&amp;alpha;-MEM</td><td>Sigma</td><td>M8042</td><td>in NK medium</td></tr></tbody></table>

high-content screening assay for the identification of antibody-dependent cellular cytotoxicity modifying compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

To demonstrate how the assay works in real life, we created a test library of 16 compounds selected randomly from the lab shelves (Figure 3). In addition, DMSO was also included as a negative control, and three microtubule polymerization inhibitor compounds (colchicine, vincristine, and podophyllotoxin) as positive controls. The latter were expected to inhibit ADCC by interfering with NK cell migration to the cancer cells and NK cell degranulation. All test compounds and DMSO were placed onto the test library plate in quadruplicates, and DMSO was also added to the first and last columns of the plate (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64485/64485fig03.jpg" /> 
Figure 3: The compound library used for testing the HCS ADCC assay. (A) The plate map of the compound library is shown. Each compound is present on the library plate in quadruplicates. DMSO was added as a negative control to the first and last column of the plate. Wells containing DMSO and the three microtubule assembly inhibitors (colchicine, vincristine, and podophyllotoxin) are highlighted in color. (B) Names, plate positions, and abbreviated names of test compounds are presented. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64485/64485fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Using our test library, we ran the assay and evaluated the results as described in the protocol. Since JIMT-1 cells express EGFP, they can be easily distinguished from the effector cells that are non-fluorescent. The assay is based on detecting the change in the number of surface adherent (viable) target cells. Under our assay conditions, NK cells caused approximately 50% JIMT-1 cell death (+NK group), while none of the test compounds caused any toxicity in the absence of NK cells (-NK group) (Figure 4). The assay conditions were previously optimized to achieve this medium level of cytotoxicity18, assuming that this would permit the identification of both ADCC activator and inhibitor compounds. The positive control microtubule assembly inhibitors showed up as &#34;hits&#34; in all quadruplicate positions as expected, indicating the high reliability of the assay (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64485/64485fig04.jpg" /> 
Figure 4: Testing a compound library in the ADCC model. (A) Images of ADCC reactions were taken 3 h after co-incubation of NK cells and JIMT-1 cells in the presence of DMSO with the 10x objective of the HCS imager. (The scale bar is 200 &#181;m.) One part of the image is magnified, showing the unstained effector NK cells and the EGFP-transduced target JIMT-1 cells. (The magnification of the original image is 4x, the scale bar is 50 &#181;m.) (B) The ADCC assay was performed with EGFP-transduced JIMT-1 breast carcinoma cell line and CD16.176 V.NK-92 cell line. The applied effector to target (E:T) ratio was 2:1. In the case of -NK group, the JIMT-1 EGFP cells were incubated without NK cells and anti-HER2 antibody, while in the +NK group (ADCC) 10 &#181;g/mL trastuzumab was added to the JIMT-1 and NK cell co-cultures. The number of viable JIMT-1-EGFP cells was detected using high content analysis equipment immediately after the addition of NK cells and after 3 h of incubation. Target cell viability in the +NK group was 50% of that in the -NK group. The red dashed line shows the threshold value, which represents samples with &#8805;70% viability compared to the average of DMSO control (n=20) killing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64485/64485fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds at JoVE.com

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

high-content-screening-assay-for-identification-antibody-dependent

Nanyang Technological University

Research

JoVE Journal

Cancer Research

1.9K Views.  University of Debrecen. Antilichaamafhankelijke cellulaire cytotoxiciteit, of kortweg ADCC, kan worden gemedieerd door natural killer-cellen, die we NK-cellen, granulocyten en de macrofagen noemen. Dit zijn de effectorcellen van ADCC. Als een antilichaam zoals trastuzumab, dat zich richt op de epidermale groeifactorreceptor, HER2, zich bindt aan tumorcellen, dan gebruiken deze effectorcellen hun FcyR-receptoren om het constante FC-gebied van de antilichamen te binden.Het antilichaam overbrugt de tumorcellen e...

Video: High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Immunology and Infection

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Using Reference Reagents to Confirm Robustness of Cytokine Release Assays for the Prediction of Monoclonal Antibody Safety

New immunostimulatory antibody drugs designed to either directly stimulate specific immune cells or indirectly enhance the immune response by blocking or activating an endogenous regulator of the immune system have the potential to cause serious immune-related adverse events such as cytokine release syndrome (CRS). It is, therefore crucial to assess the safety profile of such drugs with a combination of in vivo and in vitro experiments before first-in-human dose administration. Cytokine release assays (CRAs), where the proposed antibody therapeutic is co-cultured with human immune cells (such as peripheral blood mononuclear cells (PBMCs), whole blood, or otherwise) and the amount of inflammatory cytokine produced is measured, are critical for hazard identification. However, different labs using different control antibodies can threaten the harmonization of CRAs, and clinically relevant controls (such as TGN1412) can be difficult to source, which can lead to less accurate or reliable results or data which are difficult to compare between laboratories. The inclusion of positive and negative controls in a CRA can ensure the accuracy and reliability of the results. The National Institute for Biological Standards and Control (NIBSC) has produced a panel of lyophilized antibody controls intended for use in various CRA platforms to harmonize results across various laboratories and assay methods. A set of three different positive control antibodies include anti-CD52, anti-CD3, and anti-CD28 superagonist (SA), which are known to induce dose-dependent CRS in patients. Each antibody is provided with an isotype-matched negative control antibody. This panel of reference reagents has previously been shown to have good inter-lab reproducibility and are suitable controls to increase the confidence and robustness of safety data from a variety of CRA platforms.

The use of cytokine release assay reference reagents allows for more reproducible and standardized in vitro safety profiles of immunotherapeutic monoclonal antibodies. Here we describe how cytokine release assays can be used alongside a reference reagent panel to predict the safety of some therapeutic monoclonal antibodies.

New immunostimulatory antibody drugs designed to either directly stimulate specific immune cells or indirectly enhance the immune response by blocking or ...

Thyroid Cancer Cells as Effectors in an ADCC Bioassay for Cancer Immunotherapy

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in cancer immunotherapy. Evaluating ADCC activity in cancer cells is essential for the development and optimization of antibody-based treatments. Here, we propose a methodological approach of utilizing an ADCC bioassay kit for quantitative assessment of ADCC reaction using thyroid cancer cells as effector cells. The protocol involves the co-culture of effector cells with target cancer cells in different ratios in the presence of a therapeutic antibody. The ADCC bioassay kit used in this experiment includes the genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. Upon the binding of the surface antigen on the target cells with the antibodies and effector cells, the effector cells release luciferase, enabling quantification of cytotoxicity through measurement of luminescence signal. In contrast to conventional ADCC assays, this method proved the binding of target antigen with antibodies and effector cells, which can produce reliable results in a short period.

Author Spotlight: Optimizing Antibody-Based Cancer Treatments via Antibody-Dependent, Cell-Mediated Cytotoxicity Assay

Here, we present a protocol for an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay using an ADCC bioassay kit. This method offers a valuable tool for elucidating the ADCC mechanism and evaluating the therapeutic potential of antibodies in cancer immunotherapy.

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in ...

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in assessing their efficacy and is a prerequisite for clinical application. However, traditional cytotoxicity assays are conducted as bulk assays and do not provide detailed information about the functional heterogeneity of the CAR T cell population. In this study, we describe a double-emulsion droplet-based method that allows for large-scale co-encapsulation of single effector CAR T cells with single target cells while enabling dual quantification of both cytotoxic effector molecules from T cells and cell death of the target cell. The protocol outlines a method for the generation and purification of CD19-specific CAR T cells, followed by their co-encapsulation in droplets with the CD19+ cell line JeKo-1, along with reagents to visualize cytotoxic effector molecule secretion (Granzyme B) and cell death (using propidium iodide, PI). We demonstrate how to generate droplets containing single CAR T and target cells using a commercially available microfluidics device for generating double emulsion droplets. Additionally, we provide examples of how to assay the functional diversity of CAR T cells in droplets using standard flow cytometry equipment. Finally, we briefly describe the temporal kinetics and heterogeneity of CD19-specific CAR T-cell killing. While this method focuses on cell death following a CAR T cell attack, it is also adaptable for examining other types of T cells, cytotoxic immune cells, and effector cell functions, such as cytokine secretion.

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in ...

An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes

Source: Powell, R. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60149">Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to measure the antibody-dependent cellular phagocytosis (ADCP) by breast milk phagocytes. Fluorescently labeled microspheres conjugated to an antigen-antibody complex, are combined with a cell suspension isolated from breast milk to induce antibody-dependent cellular phagocytosis by the phagocytic leukocytes. Upon labeling the cells with antibodies against common leukocyte antigen (CD45), flow cytometry is used to measure the fluorescence intensity of microsphere-positive viable phagocytic leukocytes, indicating the level of ADCP activity.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Target Microsphere preparation

	...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

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