[SLAS 2017] CAR-T Cell Screening in Tumor Spheroids Using Corning Spheroid Microplates

[SLAS 2017] CAR-T Cell Screening in Tumor Spheroids Using Corning Spheroid Microplates
Version:
Corning Poster

File Name/Number:
2017 SLAS Conference Poster

Year:
2017

Chimeric antigen receptor (CAR)-T cells, which are engineered to recognize target cell surface antigens expressed on tumor cells, have shown promise to affect complete remission in patients with B-cell malignancies. However, applying this approach to target solid tumors has resulted in adverse effects in clinical studies, as many of the surface antigens that are upregulated in solid tumor cells, and thus chosen as targets for CAR-T cells, are also present at significant levels in normal tissues1. This can cause toxicity of the normal tissues and may result in death. Methods for testing different models of CAR-T cells in vitro can provide further insight into viable antigen targets before these models reach the clinical stage. Historically, two-dimensional (2D) cell culture models have been used in drug discovery for the development of cancer therapeutics due to their ease of use and established compatibility with high throughput screening. Recently, more elaborate, three-dimensional (3D) cell culture models have been developed, which better mimic the in vivo tumor microenvironment, to bridge the gap between successful in vitro studies and success in clinical trials. However, conventional methods for 3D cell culture are often time consuming, display increased variability and lack throughput. Corning® spheroid microplates are multiple well, cell culture microplates with opaque walls and unique clear, round well-bottom geometry that utilize Corning Ultra-Low Attachment surface coating. The coating is hydrophilic, biologically inert, and non-degradable, which enables the rapid and highly reproducible formation of a single multicellular tumor spheroid, centered in each well. To quantify cytotoxicity of tumor cells grown in spheroids, DiscoverX® KILR® Cytotoxicity assay provides a non-radioactive, dye-free method to specifically measure target cell death in a coculture. Target cells can be engineered to stably express a protein tagged with a β- gal reporter fragment that is released into media during cell death. The addition of detection reagents results in a chemiluminescent output that can be detected using a luminometer. In this study, KILR tumor cell lines were cultured in 384-well Corning spheroid microplates to form spheroids. Tumor-specific cytotoxicity was screened after treatment with ProMab Biotechnologies EGFR scFv-CD28-CD3ζ CAR-T cells, which are engineered to target EGFR with a single chain variable fragment (scFv), and contain a CD3ζ antigen recognition domain and a CD28 co-stimulatory domain. The KILR detection reagent was added and the resulting luminescence in the spheroid microplate was detected with a plate reader. When combined with the KILR luminescent assay and ProMab Biotechnologies CAR-T cells, the Corning spheroid microplate enables a high throughput screenable CAR-T cell assay that targets tumor spheroids.