High‐Throughput Image Cytometry Detection Method for CAR‐T Transduction, Cell Proliferation, and Cytotoxicity Assays

Abstract

Chimeric antigen receptor (CAR)‐T cell therapy has drawn much attention due to its recent clinical success in B‐cell malignancies. In general, the CAR‐T cell discovery process consists of CAR identification, T‐cell activation, transduction, and expansion, as well as assessment of CAR‐T cytotoxicity. The current evaluation methods for the CAR‐T discovery process can be time‐consuming, low‐throughput and requires the preparation of multiple sacrificial samples in order to produce kinetic data. In this study, we employed the use of a plate‐based image cytometer to monitor anti‐CAIX (carbonic anhydrase IX) G36 CAR‐T generation and assess its cytotoxic potency of direct and selective killing against CAIX+ SKRC‐59 human renal cell carcinoma cells. The transduction efficiency and cytotoxicity results were analyzed using image cytometry and compared directly to flow cytometry and Chromium 51 (51Cr) release assays, showing that image cytometry was comparable against these conventional methods. Image cytometry method streamlines the assays required during the CAR‐T cell discovery process by analyzing a plate of T cells from CAR‐T generation to in vitro functional assays with minimum disruption. The proposed method can reduce assay time and uses less cell samples by imaging and analyze the same plate over time without the need to sacrifice any cells. The ability to monitor kinetic data can allow additional insights into the behavior and interaction between CAR‐T and target tumor cells. © 2020 International Society for Advancement of Cytometry

Comparison of chimeric antigen receptor‐T cell‐mediated cytotoxicity assays with suspension tumor cells using plate‐based image cytometry method

“…One can clearly observe the decrease in the number of GFP‐U266 cells over time and in respect to higher E:T ratios, whereas the GFP‐NCI‐H929 target cells showed notably lower reductions. In the unmixed wells for A‐CAR and B‐CAR, multiple large clusters are formed for GFP‐U266 indicating cytotoxic activities from the CAR‐T effectors cells, which has been observed in previous publication [17], whereas GFP‐NCI‐H929 did not show much. It is critical to mix the wells prior to image cytometric analysis to accurately count individual GFP + target cells if there are formations of large clusters.…”

“…In the recent decade, plate‐based image cytometry method has been effectively utilized for high‐throughput cell‐mediated cytotoxicity assays [14–17]. The advantages of using image cytometry for CAR‐T cell‐mediated cytotoxicity assay are the ability to perform high‐throughput analysis, time‐course measurement, and visualization of cell killing in bright field and fluorescent images without utilizing a large amount of target and effector cells.…”

“…There are a variety of fluorescent labeling that can be fit‐for‐purpose when performing image‐based cell‐mediated cytotoxicity assays [13–17]. A fit‐for‐purpose assay can be selected depending on the type of tumor cells (suspension or adherent), coculture time duration (4–96 h), or E:T ratios.…”

Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model

“…The antitumor efficacy of BBζ CAR-T cells with pure CD4, CD4:CD8 ratio of 2:1, CD4:CD8 ratio of 1:1, CD4:CD8 ratio of 1:2, and pure CD8, was evaluated using image cytometry on mCardinal-expressing CAIX + ccRCC patient-derived cell line skrc-59 as described previously. 28 , 29 …”

Formulation Considerations for Autologous T Cell Drug Products

“…While medium-throughput formulation screening for T cell viability can be implemented using 96-well plates, 384-well plates may require consideration of gaseous exchange and evaporation. Moreover, 96-well plate-based image cytometry has been demonstrated to measure CAR-T cell proliferation, transduction efficiency and target cell killing, with similar accuracy to conventional methods, with the advantage of added time-course data [ 88 ]. Data sets pertaining to prospective formulations acquired for cells suspended in multi-well plates will require translation to cell processes and scales required for patient supply.…”