Application Note
A high throughput screening campaign to streamline antibody discovery
W
ith breakthroughs in molecu- lar engineering and antibody humanisation, monoclonal
antibodies (mAbs) are one of the fastest- growing classes of biopharmaceuticals for most clinical indications. mAbs are the backbone of many treatment modalities, including antibody drug conjugates, bispe- cific antibodies and CAR-T cell therapy. Many of the approved antibody drugs
engage the same target or target multiple members of a single signalling pathway. For example, multiple approved antibody drugs target the human epidermal growth factor receptor 2 (HER2), the B-cell anti- gen CD20 and the programmed cell death 1 ligand (PDL1), although the mechanism of action, antigenic epitope and antibody format differs. This highly competitive landscape incentivises drug developers to produce next-generation antibody drugs against new targets with improved mecha- nism of actions, pharmacokinetics and delivery systems. The next generation of antibody drugs
are focusing on more challenging therapeu- tic targets such as G protein-coupled recep-
cell lines that express high surface levels of the target antigens. 2. Immunising mice with multiple types of antigens, followed by testing of the immune sera for binding to target antigens. 3. Large-scale screening of hybridoma clones for antibody binding and target specificity.
tors (GPCR), necessitating high throughput tools that can run large screening cam- paigns and software capable of easily analysing large multiplexed data sets. Current antibody screening tools such as
ELISAs are single endpoint assays that only report on binding one target antigen at a time, hence they are time-consuming and utilise large amounts of target protein. This application note highlights how researchers at ModiQuest Research used the Intellicyt® iQue Screener platform to run high through- put multiplexed assays to simplify and streamline their antibody discovery work- flow while utilising minimal sample volumes. It shows how the platform was utilised
across different stages in their antibody dis- covery process: 1. Target cell line engineering to generate
Multiplexed Hybridoma Screen: 9,600 Hybridomas were tested from two different mice immunised with cells expressing target antigen. The entire screen was completed in one day. Heat maps (red) showing results for antibody binding to control cells, cells expressing target antigen and cells expressing a related but irrelevant antigen. Profile maps (blue) combine the binding results to the three cell types, showing antibodies that bind to cells expressing the target antigen, but not to cells expressing an irrelevant antigen or to negative control cells
Drug Discovery World Fall 2018
Methods Target cell line generation: Cells were trans- duced with lentivirus encoding target anti- gen, cultured under selection pressure, and sorted into 96-well plates. After cell growth, aliquots of cells were transferred to assay plates and tested for surface antigen expres- sion using a fluorescently tagged antibody specific to that antigen. 400 engineered cell clones were screened for high surface expression of target antigen within an hour. Mouse sera evaluation: Nine BALB/c mice per group were immunised with either puri- fied protein, a DNA plasmid expressing antigen, antigen expressing cells, or lipo- some embedded antigen. Antibody binding was determined employing a fluorescent detection antibody and antibody titers were measured using a duplicate eight point, two-fold dilution series of either preim- mune or immune sera. Nearly 2,500 sera samples were analysed. To ensure antibody specificity, a multiplexed approach was used where cells expressing the antigen, a related antigen or the parental cell line was encoded with different intensities of a fluo- rescent dye and mixed into the same well. Hybridoma screening: Hybridomas were generated from two mice identified by the antibody titer assays. In order to maximise the number of antibody leads identified, 9600 Hybridomas were placed into 25 384-well plates and grown for 14 days prior to analysis. Supernatants from the hybridoma clones were mixed with the three encoded cell lines used for the anti- body titer assays to measure antibody bind- ing and specificity. Download this application note to learn
more:
http://intellicyt.com/abscreeningcas- estudy.
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