Visit our booth at AACR to learn the latest on the IntelliCyt iQue® Screener PLUS and the IncuCyte® S3 Live-Cell Analysis System. These innovative systems provide specialized instrumentation, software and reagents for live-cell analysis to accelerate discovery and development of cancer therapies and provide new insight into the mechanisms of disease at a speed, depth and scale not achievable with conventional cell analysis techniques.
At the show, please visit our poster presentations.
Using the IntelliCyt iQue® Screener to simultaneously profile antibody dependent cell cytotoxicity and cytokine release of therapeutic antibodies
Presenter: John O’Rourke, Ph.D, MBA, Assay Development Manager
Session Date and Time: Tuesday, April 17, 2018 1:00PM – 5:00PM
Location: McCormick Place South, Exhibit Hall A, Poster Section 31
Poster Board Number: 11
Authors: John O’Rourke*, Caroline Weldon*, Catriona Thomson+, Ben Tyrell+, Zhaoping Liu*
*Sartorius. Albuquerque, NM.
+Sartorius Stedim Biotech, Glasgow Scotland
Therapeutic antibodies can mediate cancer cell killing through a variety of mechanisms including antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and complement dependent killing (CDC). ADCC is induced through the binding of the antibody Fc region to Fc receptors (FcRs) expressed on effector cells of the immune system. Granule exocytosis of perforin, granzyme B, the induction of Fas ligand (FasL) expression on immune cells and the release of cytokines are major mechanisms involved in targeted cancer cell killing.
Traditional approaches to screen for antibody Fc effector functions focus on ADCC using a homogenous live/dead readout, which greatly limits the contextual and correlative value of the screening data. To overcome this limitation, a multiplex screening assay profiling ADCC using multiple cell death endpoints, and quantitating secreted proteins/cytokines was developed and analyzed using Intellicyt’s iQue Screener PLUS and integrated ForeCyt® software. The iQue Screener Plus is a high throughput flow cytometry platform featuring 3 lasers with 13 fluorescent channels and can sample a 384-well plate in 20 minutes.
As a proof of concept, a small set of therapeutic antibodies directed against the same tumor antigen were used to induce immune cell-mediated killing of tumor cells using different effector to target cell ratios and a range of antibody concentrations. To demonstrate antibody specificity, antigen positive target cells, negative control target cells and effector immune cells were barcoded with different encoding dyes and included in the same well. At specific times, a small aliquot from each well was transferred to a new assay plate to run a multiplexed cell/bead mixture assay by the addition of a cocktail of fluorescent immunophenotyping antibodies, fluorescent dyes measuring unique apoptosis parameters, and a panel of QBeads for secreted protein detection. The samples were assayed on the IntelliCyt iQue Screener PLUS with simultaneous data analysis including multi-plate analysis for cell killing kinetics. Positive/negative target cells and immune cells were digitally segregated by differently encoded fluorescence. Time-dependent cell killing of antigen positive target cells were observed using the apoptosis markers mitochondria depolarization staining and cell membrane integrity, whereas little killing was measured in antigen negative cells. The antibody set showed a range of ADCC mediated killing and granule exocytosis/cytokine release suggesting differences in Fc effector functions. These results highlight the streamlined workflow on IntelliCyt iQue Screener PLUS platform to profile therapeutic antibodies in a functional ADCC/cytokine release assay predicting their clinical efficacy.
Antibody internalization assays for cancer drug discovery
Presenter: Nicola Bevan, Principal Scientist
Session Date and Time: Sunday Apr 15, 2018 1:00 PM – 5:00 PM
Location: McCormick Place South, Exhibit Hall A, Poster Section 35
Poster Board Number: 16
Authors: N. Bevan, T. Dale & D. Trezise (sponsor Daniel Martinez Molina)
Essen BioScience R&D, Welwyn Garden City, United Kingdom, AL7 3AX
Monoclonal antibodies (mAb) and antibody-drug conjugates (ADCs) are widely used as anti-cancer therapeutics. A key property of these biologics is the extent and rate of internalization into different cells, which governs their efficacy, safety and pharmacodynamic profile. Quantifying and comparing the internalization rates of different Ab drug candidates and production batches is therefore a critical step in the biopharmaceutical selection and optimization process.
Here, we describe novel and enabling cell-based Ab internalization assays that are turnkey, medium throughput and geared toward industrial biologics discovery. Internalization measurements are made over time on 96-well microplates using live-cell analysis (IncuCyte® S3) and a fluorescent pH-sensitive dye coupled antibody-binding fragment (FabFluor) that binds the test mAb Fc region using a single step, no wash labeling protocol. An increase in fluorescence signal is observed as the mAb complex is internalized into the acidic lysosome. To validate this approach trastuzumab (Herceptin, Her-2) or rituximab (Rituxan, CD20) were mixed with the hFabFluor reagent (1: 3 molar ratio, 15min), serially diluted in complete media (1:2) and added to pre-plated BT-474 or Raji cells (no wash). Cell images (10-20x) were taken and automatically analysed for fluorescence area every 30min for up to 48h. Both trastuzumab (BT-474) and rituximab (Raji) caused clear time- and concentration-dependent internalization (EC50 values 2.1 and 2.6nM, respectively). The fluorescence signal was punctate, outside of the nucleus and strongly co-localized with a lysosomal marker (LysoSensor). In line with known marker expression profiles, specific internalization of mAbs to CD45, CD71 and CD3, but not CD20, was observed in Jurkat T-lymphocytes and CD45, CD71 and CD20, but not CD3 in Raji B cells. As a proof of concept for screening and direct comparison of test mAbs, 6 commercially available CD71 (transferrin receptor) Abs were labeled with mFabFluor reagent, serially diluted (1:2, 4.6-10000 ng/ml-1) and added to HT1080 osteosarcoma cells. 3 of the Abs produced a large internalization signal with detection <50ng mL-1, whilst the other 3 were internalized weakly with signal only visible at higher concentrations. A mean Z’ value of 0.82 was calculated from control wells indicating a microplate assay with high precision and robustness.
Taken together these data support the validation of a simple, integrated and quantitative solution for directly studying internalization of mAbs into cells which can easily be scaled to compare multiple Abs in parallel. This method enables mAb and ADC internalization measurements to be implemented at early stages of the biologics discovery process and will prove valuable in efficacy, safety and pharmacokinetic optimization.
Development and optimization of matrigel-based multi-spheroid 3D tumor assays using real-time live-cell analysis
Presenter: Tim Jackson, Research Engineer
Session Date and Time: Wednesday Apr 18, 2018 8:00 AM – 12:00 PM
Location: McCormick Place South, Exhibit Hall A, Poster Section 1
Poster Board Number: 20
Authors: K. Patel1, M. Oliver1, G. Lovell1, N. Holtz2, T. Jackson2, N. Dana2, T. Dale1, D. Trezise1
1Essen BioScience Ltd, Welwyn Garden City, AL7 3AX UK & 2Essen BioScience Inc, Ann Arbor, Michigan, 48108 USA
The tumor-associated extracellular matrix (ECM) provides critical biochemical micro-environment cues, as well as an essential structural scaffold, for solid tumors to survive and grow (see Pickup et al. 2014 for review). With a view to enabling more translational and turnkey 3D in vitro assays for cancer biology, we have developed and optimized techniques for seeding, growing and automatically quantifying the properties of multiple tumor spheroids in ECMs in 96-well format using real-time live-cell analysis.
Matrigel (Corning) was dispensed across a range of volumes (20 – 50 µL) and concentrations (1 – 5 mg/ml-1) into flat-bottomed 96-well TC micro-plates to form a solidified base layer. Tumor cells (A549, MCF-7, SKOV-3, MDA-MB-231) were seeded on top (1 – 2K cells per well), and in some experiments a full ECM sandwich was created by addition of a further volume of Matrigel (2 – 25%, 0.2 – 5 mg/ml-1. Using a custom autofocusing method, phase contrast, bright-field and fluorescence images (10x) were captured every 6h for 7 days from within the cell incubator (IncuCyte S3 live-cell analysis system). Typically, 20 – 80 spheroids were analyzed in each well. All four cell types formed multiple cell aggregates within the first 3 days, ranging in diameter from 30 – 80 microns. A549, SKOV-3 and MCF-7 multi-spheroids grew as round aggregates while MDA-MB-231 spheroids displayed stellate branching characteristic of an invasive morphology. At Matrigel volumes less than 40 microlitres or concentrations less than 3 mg/ml-1, cells penetrated to the base of the plate and grew as ‘flat 2D’ cultures. Using a novel bright field image analysis algorithm, the number, area and average size of the spheroids could be computed over time non-invasively and without the use of fluorescent labels. Once formed, A549, SKOV-3, MCF-7 and MDA-MB-231 multi-spheroids increased 3.0-, 1.6-, 3.8- and 3.3-fold in size over 4 days, respectively. Treatment of A549 multi-spheroids with the DNA enzyme topoisomerase inhibitor camptothecin (1000 nM) inhibited growth with comparable spheroid size at day 0 and day 7 post treatment (average brightfield area 1.4 x 104 micron2). Using fluorescent protein reporters for apoptosis (Annexin V) and cell viability (IncuCyte CytoTox Green) we could verify camptothecin-induced cell death (fluorescence values 149±16% of control (Annexin V) and 243±51% of control (CytoTox). A concomitant decrease of stably expressed RFP (to 3±1% of control) was observed.
The combination of protocol developments, novel image acquisition/analysis algorithms and cell health reporters creates an integrated solution for measuring growth and vitality of multiple small spheroids in a relevant and 3D bio-matrix over time. This approach should be applicable to primary- and patient–derived organoid tumor samples as well as cancer cell lines.
Pickup, MW, Muow, JK, Weaver, MW (2014), EMBO Rep. 15(12): 1243–1253