Cell Linking Moieties (CLMs) are bi-specific antibody fragments that are capable of recognizing epitopes on tumor cells and specific effector cells via unique cell surface receptors. They are comprised of 2 scFv elements recognizing unique epitopes, bound by a linking element. CLMs offer a distinct advantage in that they do not rely on the expression of MHC class I molecules, generation of specific antigenic peptide, or ex vivo manipulation of cells to exert their tumor cell killing effect. To identify the proper orientation of scFv elements and to define the most potent and selective CLM, we have established an image-based screening platform capable of interrogating a large number of CLMs based on their ability to activate effectors and specifically kill tumor cells. We demonstrate the ability to screen a number of CLMs targeting wild type EGFR and HER2 receptors on fluorescently-tagged tumor cell lines for their ability to recruit CD3+ T-cells from unstimulated PBMC pools and mediate selective cell killing. CLMs were created by transient transfection of HEK293 cells with PCR-generated DNA fragments in 96 well plates. Supernatant was harvested 3 days post transfection. Dilutions of crude supernatant were applied to A549, MDA-MB-468, and SW620 (negative) target cells expressing EGFR or HER2 receptors in the presence of unstimulated PBMCs at 10:1 PBMC:Target ratio in 384 well plates. PBMC to target ratios were pre-optimized by running a titration from 2:1 through 1:30 Target:PBMCs in a kill assay. Resulting target cell loss (cell death) was compared to control wells (mock transfected supernatant). In the screening campaign, a primary screen first selects CLM hits based on killing ability, and then a secondary screen is employed to provide dose-responses, IC50s, and additional information, such as non-specific killing of negative lines. Using this image-based screen, consistent Z-scores above 0.5 were obtained, which corresponds to 12 standard deviations between the positive and negative controls, indicating an excellent dynamic range and low variability in the assay. In a proof of concept study using this platform, two EGFR-CD3 CLMs were selected from a larger list and characterized further in the secondary screen. In the top candidate, secondary screening resulted in IC50 average values of 1632 and 12,971 - fold dilution of CLM-containing HEK293 supernatant in A549 cells, and MDA-MB-468 cells, respectively. At high doses of top-candidate CLMs, no target cells remained in PBMC co-cultures, representing a near 100% killing effect. No killing was observed in the negative control line, SW620. Consistent ranking of CLM candidates was observed among 4 donors of PBMCs evaluated. In conclusion, this image-based screening platform allows for well- and cell-level killing information to be obtained with specific loss of target cells in complex mixtures to be accurately calculated without the presence of radioactive compounds like 51Cr. Furthermore, this method allows for kinetic analysis where the same well can be scanned and analyzed at multiple timepoints in the assay to understand the dynamics of killing activity. Lastly, the development of a specific and selective high throughput functional screening assay using non-terminal high content fluorescent imaging to rapidly select CLM-T cell hits results in a shortening of the developmental time line.

Citation Format: Jeffrey Rosenbloom, Jason Isaacson, Srinivas Rengarajan, Paul Szymanski, Jennifer Buenviaje, Kristi Elliott, Francois Lebel, John A. Barrett, Richard Einstein. Development of a high-throughput imaging screen for the functional assessment of cell-linking moieties using effector and target cells in a cell kill assay. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B16.