When developing cell lines for the production of antibodies, it is essential to choose healthy clones with high levels of IgG per cell. This process typically requires several assays on multiple platforms to get enough information to select optimal clones. Due to the expense and effort involved, this limits the number of clones that can be evaluated. We have developed the Cy-Clone™ PLUS Kit, a rapid, no-wash screening assay that simultaneously provides IgG concentration, IgG concentration per cell and per viable cell, cell viability, and cell density in every well of a 384-well plate. CHO cells transfected with human IgG genes were inoculated, plated by limiting dilution, and cultured for 20 days. Cells were mixed with the reagents from the Cy-Clone PLUS kit and then read on the iQue® Screener PLUS platform in ~22 minutes. Using ForeCyt® Software, readouts for each well were visualized using heat maps and Profile Map to identify wells meeting multiple IgG production criteria. The Cy-Clone PLUS kit with the iQue Screener platform provides all the relevant data needed to select and rank the best clones in a single well without screen-size limitations. The Cy-Clone PLUS kit enables you to make more informed decisions earlier, shorten development time, and decrease costs.

Monitoring the ex vivo activation of human T lymphocytes is key to developing optimized adoptive cell therapy protocols. We describe here the development of a large scale multiplexed assay using high throughput flow cytometry to daily monitor T-cell activation in PBMCs from multiple donors. On Day 0, PBMCs from multiple donors were profiled to confirm immunophenotype before and after T-cell enrichment with CD3/CD28 magnetic beads. Enriched T cells were cultured in the presence of the beads for 3 days. On Days 1, 2, and 3, samples were removed from the cultures and evaluated using assays that combined cell phenotype measurements with analysis of multiple secreted cytokines. In each well of the assay plates, secreted levels of 11 cytokines and multiple cellular readouts including the presence of cells expressing CD69, CD25 and HLA-DR over the course of a three-day activation protocol were determined. Cell viability and cell number for different subpopulations were also evaluated. Each 96-well plate contained T cells from 12 different donors, in quadruplicate, in two different dilutions. Each plate was read on the iQue® Screener PLUS system in less than 15 minutes. The results indicate time resolved, donor-to-donor differences in activation markers. These large-scale experiments with high throughput flow cytometry provide extensive T-cell activation profiles on multiple donors with rapid assay turnaround time. The use of these assays can provide valuable information for optimizing adoptive cell therapy development and manufacturing protocols.

Abstract The success of immunomodulatory approaches for the treatment of disease has generated tremendous interest in discovery of new immunotherapy based therapeutics. Immunotherapy has the potential of affecting - positively and negatively - the myriad interactions among cells and signaling molecules regulating the immune system, and projects should include high throughput screening campaigns aimed at profiling the effects of compounds on these complex interactions. We describe a no-wash, high throughput screen that provides a multiparameter activity profile for a structure activity relationship (SAR) library of immunomodulatory compounds. Seven known immunomodulatory compounds were used as templates to generate an SAR expansion library of 1438 compounds. The selection utilized a combination of various substructure and similarity search methods combined with the generation of virtual templates. Phytohemagglutinin (PHA) activated PBMCs were treated with the compounds at two doses, 10µM and 50µM. Immunophenotyping was done by α-CD3 and α-CD8 antibodies and differential toxic effects of compounds on the viability of individual cell subsets plus effects on the secretion of three cytokines were used to generate an immunological profile for each compound tested. We demonstrate the combination of a robust SAR expansion system with high throughput, multiplex screening on primary cells to generate activity profiles that can be used for identifying viable therapeutic candidates.