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 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.

Immunotherapy with anticancer antibodies, immune checkpoint inhibitors, or chimeric antigen receptor-expressing T (CAR-T) cells represents a powerful approach to cancer treatment^1,2,3. 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 cancer^4,5,6. It primarily acts by inhibiting the proliferation stimulating effect o....

NOTE: Key steps of the assay workflow are presented in Figure 2.

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 .......

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 ont.......

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 as.......

LV received funding from National Research, Development and Innovation Office grants GINOP-2.3.2-15-2016-00010 TUMORDNS", 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 György Vereb and Árpád Szöőr for their help with the use of the NK-92 cell line and for technical advice.