【資料】CAR-T開発のための高性能ソリューション（英語版）

Intellicyt® iQue3 Smarter Solutions for Next-Gen CAR-T Development Application Compendium

Contents Faster, Smarter Flow Cytometry to Advance CAR-T Discovery and Development Introduction .................................................................................................................................................................3 Rapid, Microvolume, High-Throughput Sampling Technology ........................................................3 Powerful Multiplexing Approach Through Encoding Technology .................................................3 Integrated Software Solution ..............................................................................................................................4 Conclusion: Reduce Errors and Time to Actionable Results .............................................................4 Applications of the iQue® Advanced Flow Cytometry Platform High-Throughput Cytokine Profiling in Human Peripheral Blood Mononuclear Cells .........4 Screening Ex Vivo Conditions Which Increase Memory T Cell Frequency ...............................7 Multiplex T Cell Activation Assay for Phenotypic Screening of Kinase Inhibitors ................... 9 Find out more For more information, please visit www.sartorius.com/car-t-research

Faster, Smarter Flow Cytometry to Advance CAR-T Discovery and Development Introduction Rapid, Microvolume, High-Throughput Sampling Technology Chimeric antigen receptor T cell (CAR-T)-based therapy, The iQue3 advanced flow cytometry platform has rapid, using engineered T cells to fight cancer, is a transfor- microvolume, high-throughput sampling technology mative technology like gene therapy or regenerative based on flow cytometry that delivers rich content with medicine that may forever change the landscape of sampling times of 5 minutes for a 96-well plate and medicine. CAR-T cell-based therapy uses immune under 20 minutes for a 384-well plate. An entire plate cells — either the patient’s own (autologous) or from a of data is processed at one time because samples are donor (allogeneic) — that have been genetically trans- delivered to detectors in an air-gap delimited stream. formed and are then infused into the patient to bind to The technology requires less than 10 µL, preserving cancer cells and destroy them. precious sample for downstream analysis while reducing reagent costs. The discovery and development process for CAR-T therapies has many steps, including: Powerful Multiplexing Approach Isolation of circulating T cells Through Encoding Technology Early in vitro assays deploying specific tumor cell models and understanding immune cell activation The iQue advanced flow cytometry platform performs Imaging and flow cytometry to assess biological high-throughput assays with cells and beads in sus- responses pension, enabling a powerful multiplexing approach through encoding technology. Encoding technology This application compendium demonstrates how allows screening against multiple target antigens in the you can advance and accelerate your CAR-T-based same experiment because multiple populations of cells discovery by illustrating some of the applications of the or beads bearing different antigens of interest can be Intellicyt® iQue3 advanced flow cytometry platform. combined into the wells of assay plates. This ability to simultaneously assess parameters from multiple cell types in a single well makes the iQue platform ideal for researchers delving into the complexities of the immune system, such as changes in signaling molecules and cell function. For example, it allows measurement of T cell activation by proliferation, cytokine secretion, and changes in subtype populations (immunophenotyping) in the same analysis, for deeper, more nuanced understanding of the biology. 3

Integrated Software Solution Conclusion: Reduce Errors and Time to Actionable Results The iQue advanced flow cytometry platform includes Forecyt® Software to produce actionable data by To perform an effective screening campaign, the generating plate heat maps, histograms, plots, dose screening technologies and reagents you use must have response curves, and profile maps. A multiplate analysis the speed and sensitivity to support high-throughput, feature, called Panorama, generates an analytical “big cost effective analysis, and meaningful data. The iQue picture” that automatically compares, identifies, and advanced flow cytometry platform allows you to gain ranks data across multiple plates of an experiment, as deeper insights with multiple parameter data acquisition well as multiple plates in an experiment over several for multiple cell types, expand the scope and scale of days. In addition, criteria threshold slider bars adjust data your research with fast, high-throughput flow cytometry on the fly for real-time “what if” analyses of plate data capabilities, save on reagent costs and conserve your with the click of a mouse. limited samples, and streamline your workflows with powerful, built-in data analysis and visualization tools. Forecyt Software is purpose-built to manage large data sets from microtiter plates, and includes data analysis and visualization tools that let you work with your data in real time instead of waiting for batch analysis tasks. Immediately see the impact of your actions for better, more efficient insight. Applications of the iQue® Advanced Flow Cytometry Platform High-Throughput Cytokine Profiling in Human Peripheral Blood Mononuclear Cells Immune function involves a coordinated set of secreted the ability to multiplex the detection of multiple proteins protein signals across multiple cell types, and the regu- simultaneously; however, these assay protocols are often lation of such signals depends on cytokine-dependent long and laborious. In addition, many bead-based assays cell-to-cell communication. These complex signaling require multiple wash steps, which limits the ability to pathways between cells are an important target across automate the assay and causes bead loss, increasing the drug discovery process, from primary screening to variability in the results. toxicity profiling. The Multicyt® Qbeads Plexscreen reagent leverages the Profiling of secreted proteins is ideally achieved by benefits of the iQue advanced flow cytometry platform the simultaneous detection of multiple proteins. This to provide a unique, no-wash protocol that is ideal for maximizes the contextual and correlative value of the medium- to high-throughput screening (Figure 1). data. Traditional technologies, such as plate-based en- Qbeads are assembled into user-specified multiplate kits zyme-linked immunosorbent assays (ELISAs), are limited that can contain up to 30 analytes (Figure 2). Compared because they are inherently single-endpoint readouts. to other bead-based protein detection assays, Qbeads Newer technologies, such as bead-based ELISAs, offer provide an easier screening workflow, lower cost, faster 4

Qbeads-figure1 Qbeads Assay Principles A. B. Free soluble C. protein Complete Qbead sandwich complex Fluorescently labeled Protein-specific Captured detection capture antibody protein antibody Figure 1: Qbeads function by performing a sandwich ELISA on the surface of beads. (A) Qbeads are coated with analyte-specific capture antibodies. (B) Qbeads capture analyte that binds to the analyte-specific antibodies on the bead surface. (C) A fluorescently-labeled detection antibody is added, causing the beads to fluoresce in proportion to the analyte concentration in the sample and providing a signal detectable on an iQue platform. Qbeads-figure2 Multiplexing Qbeads A. B. RL 1 Color Bead 1 Bead 2 RL 2 Color Bead 3 RL1 Figure 2: The simultaneous detection of multiple analytes is possible by using mixtures of beads specific for different analytes. (A) Beads 1, 2, and 3 are each encoded with unique intensities of two fluorescent colors, RL 1 and RL 2. Each bead is specific for one type of analyte, represented by the different shapes. (B) After the beads are run through the iQue platform, they are categorized by their fluorescence signature, which allows the binding signals for the appropriate analytes to be separated in a multiplexed sample. plate read times, and lower sample volume requirements. Methods These advantages allow the practical screening of large On three consecutive days, cryopreserved PBMCs libraries for the ability to modulate secreted protein from a single donor were thawed and treated in 384- profiles. well plates with known T cell stimulating agents. The T cell stimulation methods included: (1) co-treatment Here, we present a case study using Qbeads to profile with CD3 and CD28 antibodies (top dose = 5 μg/mL for cytokine secretion in human peripheral blood mononu- each antibody, (2) co-treatment with PMA (top dose = clear cells (PBMCs). We performed a multi-day study to 10 ng/mL) and ionomycin (top dose = 10 μg/mL), and examine cytokine secretion in a 7-plex assay representing (3) treatment with Phytohemagglutinin (PHA; top dose activation of Th1/Th2/Th17 T helper cells. = 5 μg/mL). The secretion of IL-2, IL-4, IL-6, IL-10, IL-17A, IFNγ, and TNFα were detected and quantified directly Our results highlight the turnkey solution represented by from the supernatants. the Qbeads reagents on the iQue platform, and illumi- nate the tremendous value of multiplexing, not only in time and cost savings, but also in the ability to generate correlative data in a physiologically relevant context. 5 RL2

Results Assay miniaturization possible on the iQue platform We observed different cytokine secretion profiles allowed a cost effective screen of a dose response depending on which PBMC stimulation method we for PBMC stimulation, and revealed PHA as a weak used. A subset of the cytokines assayed (IL-4, IL-10, IFNγ inducer rather than a negative treatment, thus and IFNγ) highlights some of the most significant eliminating the need for time-consuming dose-finding differences between the three stimulation methods. experiments. PMA/Ionomycin stimulation caused no observable IL-4 or IL-10 secretion at any dose but induced a strong IFNγ The Multicyt Qbeads portfolio presents an opportunity response (Figure 3). CD3/CD28 and PHA produced to elevate the screening of secreted proteins to an a similar cytokine profile — IL-4 is negative at all doses unprecedented level of efficiency by offering a stream- while both IL-10 and IFNγ are positive. lined, multiplexed workflow at significantly lower cost than many existing technologies. Summary IFNγ secretion across the three stimulation methods References emphasizes the value of screening in a dose response Narang, R., et al. Application of Multicyt Qbeads for series. Of the three stimulation methods, CD3/CD28 was High Throughput Cytokine Profiling in Human Periph- the most potent IFNγ inducer, followed by PMA/Iono- eral Blood Mononuclear Cells. Sartorius Application mycin, then PHA. Interestingly, if we had screened PHA Note, 2014. in a single-dose format, with a dose in the middle of the range tested here, it would have likely scored negative for IFNγ secretion. PMA/Ionomycin CD3/CD28 PHA 40000 30000 IL-4 20000 MFI 10000 Limit of Detection 40000 30000 IL-10 MFI 20000 10000 Limit of Detection 0 300000 200000 IFNγ MFI 100000 Limit of Detection 0 Dose Dose Dose Figure 3: Using Qbeads shows different cytokine profiles for three different PBMC stimulation methods. The dotted line represents 3 standard deviations of the mean response for unstimulated controls (N=96). All dose response series are 1:2 serial titrations. 6 Day 1 Cytokine Profiles for 3 Different PBMC Stimulation Methods

Screening Ex Vivo Conditions Which Increase Memory T Cell Frequency The ex vivo expansion of T cells is a critical process in Methods the biomanufacturing of adoptive cell therapies, such as We performed screening studies using human chimeric antigen receptor (CAR) T and tumor infiltrating peripheral blood mononuclear cells (PBMCs) from lymphocyte (TIL) therapies. Recent clinical studies show three donors cultured in a single, 96-well plate over that a correlation between the persistence of subsets of three days and using 14 different culture conditions. functional memory T cells, including T memory stem cells In each assay well, we distinguished live immune (Tscm), central memory T cells (Tcm) and other less differen- cells from dead cells by staining with a fluorescent tiated T cell subsets, is responsible for long term anti-tumor membrane integrity dye that only enters dead cells or responses in patient outcomes. This suggests ex vivo T cell those with a compromised membrane and staining expansion protocols generating higher percentages of the nucleic DNA by intercalation. Tscm and Tcm in the T cell product are critical to significant clinical improvements in adoptive cell therapies. We immunophenotyped live cells by staining with a fluorescent antibody panel to separate CD3+ (T cells), Here, we show results from a robust T memory cell and CD3- (non-T cells), CD4+ (T helper cells) and CD8+ cytokine profiling assay optimized for 96- or 384-well (T cytotoxic cells). The antibody panel also included microtiter plates that runs on the iQue advanced flow five different T cell surface markers for T naive/memory/ cytometry platform. The Intellicyt® T Cell Memory Cell effector cell phenotyping: CD45RA, CD45RO, CD27, and Cytokine Profiling Assay Kit (TCA Kit) was developed CD62L, and CD95. Effector cytokines secreted by to address the need to monitor T cell phenotype and ex vivo expanded T cells, including pro-inflammatory function for improved ex vivo expansion protocols where cytokine IFNγ and anti-inflammatory cytokine IL-10, profiling of memory subsets is crucial. This assay includes were measured in a sandwich immune assay format by antibody markers to identify T cells and identifies naive, two different Qbeads, included in the same well (see Tscm, Tcm and TEFF. In addition, a functional analysis Figure 4). We analyzed T cell expansion time course data of the cells is performed by quantitating the levels of and produced the series using the Forecyt software secreted IFN and IL-10 simultaneously with phenotypic package. measurements. One Well Self-Renewal Potency Cd3+ T Cells Cd4+ T Helper TN TSCM TCM TTM TEM TEMRA TTE Activation CD45RA: + + - - - + + CD45RO: - - + + + + - CD27: + + + + - - - Cd8+ T Cytotoxic CD62L: + + + - - - - CD95: - + + + + + + Cytokine Secretion Live Dead Pro-Inflammatory Anti-Inflammatory Cytokine IFNγ Cytokine IL-10 Cell Membrane Integrity Dye IFNγ IL-10 Capture Capture Bead Bead Cytokine Detection On Qbeads® Figure 4: Schematic of this multiplex assay design. Multiplexed measurement in each well. 7

Results increased in the wells treated with a cocktail containing Figure 5 shows the results from Donor 1 on Day 5. Tcm IFNß compared to the well treated with CD3/CD28 (especially cytotoxic CD8+ Tcm) increased in frequency Dynabeads alone. CD3+ T cell counts in culture supple- in six cocktails containing IFNß, and Tscm (particularly mented with cytokine cocktails containing IFNß also helper CD4+ Tscm) increased in frequency increased in slowed down cell proliferation by approximately 50% four cocktails containing IL-21 (Figure 5A). In Figure 5B, (Figure 5C). the 2D plots (CD27 vs. CD62L) show Tcm cell frequency A. B. Tcm Tscm Tcm Comparison Donor 1 Donor 1 Treatment 2 Treatment 7 5.0 5.0 (CD3/CD28 (CD3/CD28Dynabeads IL-21 Dynabeads only) + IL4/IL7/IFNβ) 4.0 4.0 BO1 & RA-RO+CD95+ GO1 & RA-RO+CD95+ 3.0 3.0 2 9 11 14 107 107 106 Tcm 106 Tcm 2.0 2.0 105 105 2 104 104 1.0 1.0 -105 Tem Ttm -105 Tem Ttm 0.0 0.0 -104 -104 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 -104 -105 104 105 107 -104 -105 104 105 107 16 Treatments 16 Treatments CD27 (RL2-H) CD27 (RL2-H) Donor 1 Donor 1 5.0 5.0 C. IFNβ 4.0 4.0 CD3+ T Cell Count 7 10 12 13 15 16 3.0 3.0 Donor 1 2.0 2.0 2.0 1.8 2 2 1.6 1.0 1.0 1.4 1.2 6 9 11 14 0.0 0.0 1.0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0.8 16 Treatments 16 Treatments 0.6 0.4 0.2 7 10 12 13 15 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0.0 0 2 4 6 8 10 12 14 16 CD3|CD28 - + + + + + 16 Treatments Dynabeads IL-4 | IL-7 - - + + + + IL-6 - - - + - - - + + + - - - + + - IL-15 - - - - + - - + - - + + - + + + IL-21 - - - - - + - - + - + - + + - + IFNβ - - - - - - + - - + - + + - + + 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 CD3|CD28 - + + + + + Dynabeads IL-4 | IL-7 - - + + + + IL-6 - - - + - - - + + + - - - + + - IL-15 - - - - + - - + - - + + - + + + IL-21 - - - - - + - - + - + - + + - + IFNβ - - - - - - + - - + - + + - + + Figure 5: Increased T memory cell frequency after ex vivo expansion in cytokine supplemented media. Cytokine cocktails containing IFNβ or IL-21 increase the T central memory cell frequency or T stem cell-like memory cell frequency, respectively. Example of Donor 1 on Day 5 was shown here. (A) T central memory cells (Tcm), especially CD8+ Tcm showed frequency increase in 6 cocktails containing IFNβ; T stem cell-like memory cells (Tscm), especially CD4+ Tscm showed frequency increase in 4 cocktails containing IL-21. (B) The well-scan of 2D plots (CD27 vs. CD62L) showed Tcm cell frequency increase in the well treated with a cocktail containing with IFNβ, compared with the well treated with CD3 | CD28 Dynabeads. (C) CD3+ T cell counts showed cocktails with IFNβ slowed down the cell proliferation about 50%. 8 CD8+ CD4+ CD8 Tcm (Folds of Donor 1_CD3/CD28) CD4 Tcm (Folds of Donor 1_CD3/CD28) CD8 Tscm (Folds of Donor 1_CD3/28) CD4 Tscm (Folds of Donor 1_CD3/28) CD3 Cell Count (Folds of CD62L (BL1-H) Donor 1_CD3/28) CD62L (BL1-H)

Summary Multiplexed cell and secreted cytokine measurements Here we showed that media supplements had in a single assay offers an improvement over common profound effects on the final T memory subset, immunology research workflows that require multiple cellular composition, and cytokine release. Despite assays run on different platforms. The kit allows spatial the complexity of T cell biology, the simple assay and temporal analysis of T memory cell phenotypes and analysis workflow of the Intellicyt® Human T and functions at different stages, all in a single high- Cell Memory and Cytokine Profiling Kit could be content, miniaturized assay that saves precious samples, useful for adoptive T cell therapy workflows. The kit decreases reagent costs, and enhances data integrity. is designed for ease-of-use by combining cells and beads, which allows multiplexing measurements References from each assay well. Liu, Z., et al. An Optimized, Multiplexed Assay for Screening Ex Vivo Conditions which Increase Memory T Cell Frequency. Sartorius Application Note, 2019. Multiplex T Cell Activation Assay for Phenotypic Screening of Kinase Inhibitors Activation of the T cell receptor (TCR) pathway in naive peripheral blood mononuclear cells (PBMCs) stimulated and effector T cells leads to T cell activation, proliferation, with anti-CD3/CD28 beads. We acquired samples on and cytokine production. Modulating TCR engagement the iQue platform and assessed cell proliferation in and signaling pathways using biologics, small molecules, viable CD4 and CD8 lymphocytes, as well as early/late or genetic engineering is highly relevant to many thera- activation markers CD69, CD25 and HLA-DR. To assess peutic areas, including cancer immunotherapy, adoptive T cell function, we quantified the levels of secreted IFNγ cell therapy, and vaccine development. and TNFα. We analyzed data and generated heat maps, IC50 curves, and cytokine levels using Forecyt software. Perturbations leading to increased hyper-responsive We used Profile Maps, a unique analysis tool of Forecyt TCR signaling and enhanced T cell activation is a to integrate assay metrics with Boolean logic to quickly major cause of autoimmune disease. Genetic defects, locate hits using defined multiplexed criteria. mutations, and other mechanisms resulting in increased T cell kinase activity are involved in many autoimmune Results pathologies, making them attractive targets for the The TCA kit can generate data on 15 different param- direct inhibition of T cell activation. eters, but as an example, Figure 6 demonstrates a plate-level analysis showing the percentage of viable The development of drugs and therapies regulating CD4 T cells that express the early activation marker TCR activity requires assays to profile T cell function and CD69 from Plate 1. Using this visualization tool, we can health. Here, we report on an optimized, high-content, quickly identify compounds that have inhibited expres- multiplexed assay using high-throughput flow cytometry sion of CD69 (CD69+ cells are in the rectangular gate), to measure T cell activation. as well as KI that drastically reduced CD4+ T cell viability (wells with no cells, for instance, well H2). These studies demonstrate the insight provided by the use of the iQue advanced flow cytometry platform for The plate-level view shows examples of inhibitors that phenotypic screening of small molecules affecting T cell affect different kinase families. In wells containing media activation. alone, 86% of the CD4 cells are activated, as assessed by CD69 expression and treatment with the FDA-approved Methods Jak 1/2 inhibitor Ruxolitinib, which reduced the percent- We used the Intellicyt® Human T Cell Activation Cell age of CD4+CD69+ cells down to 55%. The Src inhibitor and Cytokine Profiling Kit (TCA Kit) to screen a 152 PP2 dramatically inhibited CD69 expression as only 3% small molecule library of kinase inhibitors (KI) for their of the CD4 T cells expressed CD69. Other compounds ability to inhibit human primary T cell activation in showed a range of CD69 inhibition. 9

1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Negative Positive Side Scatter (SSC-H) Well-A1 Well-A4 Well-B5 Well-B10 Well-D7 Well-E10 A01 and CD A04 and CD4+ B05 and CD4+ B10 and CD4+ D07 and CD4+ E10 and CD4+ Media only Doramapimod AS-703026 Ruxolitinib JAK 1/2 PP2 Src Kinase KN-93 CaMKII p38MAPK MARK/ERK Kinase 07 07 07 07 07 07 CD4+CD69+ CD4+CD69+ CD4+CD69+ CD4+CD69+ CD4+CD69+ CD4+CD69+ 05 85.54% 05 52.53% 05 37.3% 05 54.87% 05 3.26% 05 67.09% 04 04 04 04 04 04 04 04 04 04 04 04 03 03 03 03 03 -104 -103 104 105 106 107 -104 -103 104 105 106 107 -104 -103 104 105 106 107 -104 -103 104 105 106 107 -104 -103 104 105 106 107 03 -104 -103 104 105 106 107 CD69 Figure 6: Compounds that inhibit CD69 expression. A plate-level view (from Plate 1) of CD69 expression in T helper cells generated in Forecyt software. The CD69 expressing cells are found in the rectangular gate. Wells highlighted in red are shown below with the indicated KI and the % of CD69 CD4+ cells found in the inset of each dot blot. To integrate the data for all 15 activation metrics, and cytokine secretion (wells highlighted in yellow in we used the Forecyt Profile Map data tool in the Figure 7). The overlay line graph in Figure 7 ranks all Panorama feature (see Figure 7). The left panel of the hits and the level of inhibition, providing easy shows the 11 metrics from this screening study and visualization for each metric. For example, treatment the user-defined threshold for each of these. Using with some KI completely inhibits all activation metrics, the Profile Map, we identified 27 different KI that whereas other compounds have a greater impact on inhibited expression of all T cell activation markers specific phenotypic markers or cytokines. 10

A. B. Criteria Plate 1 Plate 2 INFγ+ (pg/mL) <= 1,000.00 TNFα (pg/mL) <= 1,000.00 % live cells >= 40.0 CD4+CD69+ as % of CD4+ <= 70.0 CD8+CD69+ as % of CD8+ <= 70.0 C. Hits Ranking 100 CD4+CD25+ as % of CD4+ <= 70.0 90 80 CD8+CD25+ as % of CD8+ <= 70.0 70 60 CD4+HLA-DR+ as % of CD4+ <= 30.0 50 40 30 CD8+HLA-DR+ as % of CD8+ <= 30.0 20 10 Count of CD4+ >= 140.0 0 Count of CD8+ >= 140.0 Plate ID and Well ID Figure 7: Data generated using the unique Profile Map function of Forecyt Software. To identify hits that inhibited all T cell activation metric, we generated Profile Maps using the Panorama feature in Forecyt. The user-defined threshold level of each of the desired metrics is shown in (A), while the specific hits from the 2 plates are shaded in yellow (B). We generated a line graph ranking the hits based on the % of CD69 CD4+ cells (red line) and overlaid line graphs for 5 additional metrics for each of the hits (C). These visualization tools provide easy insight into how the various KIs impact different T cell activation metrics. Summary References The Intellicyt® TCA kit can be used as a primary or Liu. Z., et al. A kinase inhibitor phenotypic screen using secondary phenotypic screen in both the biologics and a novel multiplex T cell activation assay. Sartorius small molecule workflows. Additionally, the TCA kit is Application Note, 2019. used for functional studies in the immuno-oncology space and for characterizing T cells during cell manu- facturing processes. The TCA kit is applicable to many functional assay workflows including development of checkpoint inhibitors and cell therapies and during cell manufacturing. 11 Percentage (%) Plate 1-H08 Plate 1-B08 Plate 2-B08 Plate 1-C04 Plate 2-E11 Plate 1-D07 Plate 1-D09 Plate 2-G07 Plate 1-D10 Plate 1-E08 Plate 1-B05 Plate 1-G05 Plate 1-E07 Plate 2-G11 Plate 1-G06 Plate 1-B04 Plate 1-A03 Plate 1-A04 Plate 1-A10 Plate 1-B10 Plate 1-C03 Plate 1-F10 Plate 1-E10 Plate 2-B10 Plate 1-D11

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