CAR T cell and other adoptive cell therapy workflows

From isolation and activation to 3D cellular assays

Adoptive cell therapy (ACT) has emerged as an effective approach in immuno-oncology research. In recent years, personalized cancer immunotherapy in the form of chimeric antigen receptors (CARs) in T cells (CAR T cells) has effectively treated some forms of leukemia and lymphoma. Thermo Fisher Scientific offers protein and cell analysis tools to support researchers in developing workflows for engineering T cells or other cancer fighting cells such as natural killer (NK) cells with particular antibody-based chimeric antigen receptors.

Recommended tools for CAR cell generation

In early clinical studies (1980s), tumor infiltrating lymphocytes (TILs) were isolated from patients, expanded in culture, and re-implanted with the goal to improve the patient’s immune response.[1] This autologous method was shown to be effective at improving tumor regression in patients with metastatic melanoma.[1] Since then, adoptive cell therapy has been combined with genetic engineering to bolster T cell effectiveness and mount a stronger cancer-killing response. The workflow below highlights some of the recommended tools for CAR generation, validating their molecular properties, and assaying their effectiveness in different models.

Chimeric antigen receptor (CAR) cell workflow

Illustration of the basic CAR steps from T cell purification to cell assays and cell model testing - step 1 Illustration of the basic CAR steps from T cell purification to cell assays and cell model testing - step 2 Illustration of the basic CAR steps from T cell purification to cell assays and cell model testing - step 3 Illustration of the basic CAR steps from T cell purification to cell assays and cell model testing - step 4 Illustration of the basic CAR steps from T cell purification to cell assays and cell model testing - step 5
Dynabeads magnetic beads to isolate and activate T cells
Deliver CAR plasmid with viral vectors or CRISPR/Cas9
Co-culture and analyze for cell killing and cytokine secretion
Co-culture and analyze for infiltration, cell killing, and cytokine secretion
Infuse into model and monitor
Illustration showing T cell capture by Dynabeads magnetic beads

T cell isolation

The process of CAR cell engineering and therapy starts and ends with the subject. A blood sample is taken is taken from the subject and leukocytes are separated from other non-leukocyte populations, through a process called leukapheresis.[2,3] Mononuclear cells can then be purified using density gradient centrifugation and analyzed for cell viability and expression of immune effector-cell markers prior to activation and expansion for downstream cell engineering. In one study performed by Dr. Charles Prussak, donor-purified lymphocytes were analyzed on the Attune NxT Flow Cytometer to determine the relative CD4 and CD8 T cell populations (Figure 1).

4 panel flow cytometry scatter plots showing event acquisition and gating to reveal CD4- and CD8-expressing cells
 Click to enlarge

Figure 1. Flow cytometry analysis of purified peripheral mononuclear blood cells (PBMCs). Isolated PBMCs were analyzed by the Attune NxT Flow Cytometer for viability and CD4 and CD8 T cell populations. In the first plot (upper left), forward and side scatter channels clearly demonstrate a mixture of lymphocytic and non-lymphocyte populations based on size and granularity. Gating was first performed on the lymphocyte-sized population and analyzed for cell death based on the presence of positively stained cells (top right). Further analysis for CD3 (bottom left), CD4, and CD8 expressing cells (bottom right) revealed an expected 2:1 ratio of CD4 to CD8 cells.

Immune cell activation

Immune cells within isolated lymphocyte populations require activation and expansion prior to genetic modifications. For this purpose, it is critical to mimic antigen presentation during immune cell culture. For T cell activation, magnetic beads bound to CD3 and CD8 monoclonal antibodies, combined with activating cytokines, such as IL2, IL7 or IL15, are an effective way to mimic B cell antigen presentation and to achieve activation. This can be performed with Dynabeads CD3/CD28 in Gibco CTS OpTmizer T Cell Expansion SFM. Following incubation, as seen in Figure 2, the population then can be analyzed for proliferation and activation markers such as HLA DR, CD22, or CD69. This is an important step prior to transduction and genetic modification of cells, as over activation can cause activation-induced death. Additional analysis can be performed for cell size, viability, and proliferation, as shown in Figure 3, with flow cytometry or microscopy analysis. Indicators of health and cell function during expansion are important factors to evaluate for many downstream immunological research application areas.

4 panel flow cytometry histogram showing an increase in the number of HLA DR-expressing cells
 Click to enlarge

Figure 2. Flow cytometry analysis of activated T cells prior to transduction. Incubation and activation were performed over a 72 hr time course using magnetic CD3/CD28 activation beads. Following activation, analysis was performed on CD4- and CD8-positive T cells using Attune NxT Flow Cytometer. There is increased expression of lymphocyte populations based on activation associated antigen HLA DR compared to the 72 hr population (bottom row) with the initial time point of 0 hours (top row).

Flow cytometry histogram of the number of cells as a function of cell size in activated and non-activated T cells
 Click to enlarge

Figure 3. Quantification of T cell viability and concentration after activation. Isolated human T cells were activated using Dynabeads CD3/CD28 Untouched Human T Cell Kit in Gibco CTS OpTmizer T Cell Expansion SFM. Cells were stained with Invitrogen Trypan Blue Stain (0.4%) prior to analysis on the Countess II FL Automated Cell Counter. Beads within the sample can be gated out based on size so that only the T cell population is part of the downstream analysis. As expected, activated T cell populations appeared greater in size than non-activated T cells.

Related product areas Application
T cell product selection guide Find culturing and activation products for cell therapy
Growth factors for cell therapy products Find the appropriate growth factor for T cells
Automated cell counters For cell counting, viability and size determination
T cell expansion in clinical research Isolate, activate and expand T cells

Additional resources for T cell isolation and activation

Cancer research webinar

Presented by:
Charles Prussak, Pharm D, PhD, UCSD Moores Cancer Center

Title:
The Complex Pharmacology of T-Cell CARs

Broadcast dates:
Dec 12 & 13, 2017

Read the press release:
First FDA-approved cell therapy for leukemia utilizes Thermo Fisher Scientific’s CTS Dynabeads Technology

TOP

illustration showing CAR T genetic modification

CAR T cell genetic modification approaches

Classical retroviral or lentiviral transduction is typically used for CAR cell generation,[4,5] although there is an increased interest in pursuing the use of CRISPR/Cas9 gene editing procedures for adoptive cell transfer immunotherapies. New tumor-specific targets of interest identified through biomarker discovery efforts are under investigation by the research and translational community. For instance, Dr. Prussak’s lab is interested in ROR1, a protein normally found in the second trimester of fetal nervous system development is now being identified in some hematological or solid tumor cancers, making it an ideal target for immune therapy. To target this protein, viral particles containing the ROR1 antigen in the context of a CAR construct, were transduced into the activated T cells from Figure 3. Flow cytometry analysis was performed to determine viral infection efficiency by analyzing CD4- and CD8-positive T cells for ROR1 expression at the cell surface (Figure 4).

PBMC transduction results - control
 Click images to enlarge
PBMC transduction results - moi 1
PBMC transduction results - moi 3

Figure 4. Lentiviral transduction of viral particles carrying ROR1 CAR. Donor PBMCs were isolated and activated prior to transduction. ROR1 CAR–containing viral particles were used to transduce cells at multiplicities of infection (MOI) of 1 and 3. CD4- and CD8-positive cells were labeled with anti-ROR1 antibody recognizing the single chain variable fragment (scFv), and analyzed for positive fluorescence on the Attune NxT Flow Cytometer.

Related product areas Application
CRISPR/Cas9 Gene editing
Lentiviral production systems Cell engineering
Cell viability dyes Cell death assays for flow cytometry
CellTrace dyes Cell proliferation for flow cytometry

CAR T cell genetic assessment

Process quality control and evaluation during CAR development is necessary in both basic and translational settings to ensure efficacy of the engineered effector cell. Phenotypic assessment using antibody labeling and function-based approaches is needed, but genetic diversity and the clonal distribution of the T cell are variables that can lead to reduced efficacy and must also be evaluated. Next-generation sequencing (NGS) analysis with the Oncomine TCR Beta-LR Assay can be used at all stages of the CAR workflow to assess evenness, a measurement of a population’s T cell receptor (TCR) distribution within the T cell repertoire.

Related product areas Application
Oncomine TCR Beta-LR Assay Measure the TCRβ repertoire
Ion GeneStudio S5 systems Next-generation sequencing (NGS)

Additional resources for CAR T cell genetic engineering

TOP

Illustration of CAR T cell-mediated killing

CAR T cell killing assays

Researchers can study CAR T–mediated cell killing in cell co-culturing assays, with both 2D and 3D models. Sourcing the appropriate cells that mimic the disease models of interest can aid in the study of the effectiveness and specificity of a generated CAR model. The ROR1 CAR T cell line from Figure 4 was used in a mixed cell culture to validate its ability to kill a ROR1-positive B cell lymphocyte cell line called Jeko. To do so, ROR1 CAR T cells were incubated with Jeko cells labeled for identification and for tracking proliferation and were analyzed at 1 and 43 hours (Figure 5). At the 43-hour time point, gating on cells positive for CellTrace Yellow dye shows a significant reduction of Jeko cells compared to control (43% to 5.5%), indicating T cell–mediated killing of the Jeko cell line. However, it cannot be determined from this data whether or not the killing was achieved specifically through ROR1 binding to its target.

Flow cytometry scatter plots and histograms showing Jeko lymphoma cell death following exposure to ROR1-engineered CAR T cells
 Click to enlarge

Figure 5. CAR T cell killing of Jeko cells. Engineered ROR1 CAR T cells were incubated at a 1:1 ratio with Jeko B cell lymphoma cancer cell line. Jeko cells were stained with CellTrace Yellow dye prior to incubation and analyzed for expression at different time points. Analysis at 1 hour post incubation shows 45% of cells detected in the yellow channel on Attune NxT Flow Cytometer. After 43 hours, the analysis was performed again and only 5.5% of cells exhibited yellow fluorescence. Loss of fluorescence here indicates that the ROR1 CAR T cell killed Jeko cells.

Assessing engineered CAR T cell targeting

In a subsequent experiment using a similar co-culturing assay, two separate dyes were used to label Mec 1 cells that were engineered to express ROR1 as well as a population that remained un-altered. ROR1 CAR T cells were co-cultured at a ratio of 3:1:1 with ROR1-positive Mec cells and ROR1-negative Mec1 cells, respectively. As seen by flow cytometry analysis, ROR1 CAR T cells can differentially kill the target-expressing cells (Figure 6). When comparing all co-culturing experiments (Figure 5 and 6) it can be determined that this process is both dose and time dependent (Figure 7). It is important to note that if the ratio of CAR T cell to ROR1-negative Mec cells is increased to 10:1, a large percentage of Mec cells killed as an off-target effect is observed. Conversely with some non-CAR T cells there is also killing of the cells in co-culturing experiments, further demonstrating the subtilties of T cell killing and off-target effects. Off-target killing remains a problem for both autologous and allogeneic, donor-derived, CAR T cells and is a critical hurdle on the path to approval and effective administration of CAR-T cell products.[6] Ultimately, these are valuable assays to demonstrate T cell killing specificity and efficacy prior to moving into more biologically complex models.

Flow cytometry results indicate that the ROR1-expressing CAR T line is able to kill ROR1-positive Mec 1 cells
 Click to enlarge
Flow cytometry results indicate that the ROR1-expressing CAR T line is able to kill ROR1-positive Mec 1 cells

Figure 6. Co-culturing of ROR1 CAR T cells with Mec 1 leukemic cell line to show target specificity. ROR1 CAR T cell line were co-cultured at a ratio of 3:1:1 with ROR1 positive Mec1 cells and ROR1 negative Mec1 cells labeled with either Invitrogen CellTrace Red or Invitrogen CellTrace Yellow, respectively. Flow cytometry plots (top row) demonstrate gating to separate out live single cells. Initial gating was performed using forward and side scatter channels to separate background noise from cells. A viability dye was used to separate dead cells from the population, followed by single-cell gating. Finally, red and yellow channels were used to analyze the population for the relative amounts of Mec 1 positive and negative cells (bottom row). Analysis was compared at 1-hour (top group) and 20-hour (bottom group) post incubation on the Invitrogen Attune NxT Flow Cytometer.

Quantification of co-culturing experiments
 Click to enlarge

Figure 7. Quantification of co-culturing experiments from Figures 5 and 6. Each co-culturing scenario was quantitated and compared in a dose- and time-dependent manner to determine the effect of ROR1 CAR T cells. When the data is normalized there is an increased reduction of JeKo cells in the presence of ROR1-CAR T cells in a time- and dose-dependent manner. Similarly, there is large reduction of ROR1-positive Mec1 cells labeled with CellTrace Red, when compared to ROR1 negative Mec1 cells. This indicates ROR1-targeted CAR T cells are able to target and specifically kill ROR1-expressing Mec 1 cells in a time- and dose-dependent manner.

Related product areas Application
Cell viability dyes Cell death assays for flow cytometry
CellTrace dyes Cell proliferation for flow cytometry
Antibodies Primary antibodies recognize specific cellular markers and can serve as useful tools to characterize cell lines.

Additional resources for functional assays for CAR T cells

TOP

Illustration of CAR T cell-mediated killing

Assays for T cell killing using 3D models

3D models in the context of cancer (e.g., tumor spheroids or tumoroids) have demonstrated more biological relevancy than traditional 2D cell cultures, as they provide a context that more closely resembles the tumor microenvironment. 3D tumor models are being used more frequently in combination with adoptive cell therapy approaches. Researchers are better able to understand effector cell attributes like invasion, potency, oxidative stress, and apoptosis in the context of 3D tumor models. For instance, Figure 8 represents T cell killing through apoptosis of lung cancer spheroids. In this experiment, T cells are labeled with a far-red–fluorescent cell tracer, and spher¬oids are labeled with an apoptosis indicator. As the T cell dose increases, an increasing number of A549 cells in a lung cancer spheroid undergo apoptosis, which was quantified using the mean signal intensities of cells within the spheroid.

Figure 8. T cell–dependent killing of lung cancer spheroids. T cells were isolated from human peripheral blood and expanded for 5 days with Gibco Dynabeads Human T-Activator CD3/CD28 in Gibco CTS OpTmizer T Cell Expansion SFM. Cells were analyzed with the Invitrogen Countess II FL Automated Cell Counter to determine cell viability and concentration, and cell concentration was adjusted to 1 x 106 cells/mL in Gibco PBS, pH 7.2. T cells were then labeled with 2 μM Invitrogen CellTracker Deep Red Dye (purple) for 15 min and washed with CTS OpTmizer medium. For spheroid formation, A549 (adenocarcinomic human alveolar basal epithelial) cells were plated in a Thermo Scientific Nunclon Sphera 96-well microplate at a density of 7,500 cells/well, incubated overnight in a cell culture incubator with 5% CO2 at 37°C, and analyzed on the Invitrogen EVOS XL Core Imaging System to confirm spheroid formation. Invitrogen CellEvent Caspase-3/7 Green Detection Reagent (2 μM final concentration) (green) and the indicated number of labeled T cells were added to each well, and the mixture was incubated for 4 hr at 37°C. (A) Two examples of the response of a lung cancer spheroid to 4 different T cell concentrations are shown, each imaged on the Thermo Scientific CellInsight CX7 LZR HCA Platform using confocal mode with 10 μm Z slicing. (B) Apoptosis in A549 spheroids and the T cell dose response were quantified using the CellInsight CX7 LZR platform and Thermo Scientific HCS Studio Cell Analysis Software. The spheroids were segmented as single objects based on the brightfield image using HCS Studio software, and cells were counted within the object. Mean signal intensities of cells within the spheroid were used to quantify immune cell cytotoxicity.

If available, these models can provide a relevant context for subject-specific responses and can contribute to advances in the field of personalized cancer immunotherapy. CAR T cells engineered with a subject-specific tumor antigen receptor molecule and then co-cultured with cancer spheroids can provide a context for assessment of tumor invasion and killing by the T cell. As proof of concept, Figure 9 demonstrates that by increasing the ratio of CAR T cells to tumor spheroids there is a clear destruction and lysing of the cultured spheroid. This experiment combines a broad range of products that enables an elegant demonstration of visualizing cell-to-cell interactions between engineered immune cells and tumor spheroids.

Fluorescence microscopy images of cell lysis in cancer spheroids following exposure to CAR T cells

Figure 9. Chimeric antigen receptor (CAR) T cell invasion into cancer spheroids. HCC827 spheroids were formed using spheroid microplates for 48 hr. Then, 24 hr after the addition of EGFR scFv-CD28-CD3ε CAR T cells (ProMab Biotechnologies), spheroids were immunostained for cytokeratin-7 (green) and CD3ε (red), and counterstained with Hoechst dye (blue). As the effector-to-target ratio is increased from 10:1 (middle panel) to 40:1 (right panel), invasion of the CAR T cells into the HCC827 tumor spheroid and subsequent tumor cell lysis are visible. Images were obtained on the Thermo Scientific CellInsight CX7 High-Content Analysis Platform in confocal mode with a 10x objective, and used with permission from Corning Inc.

Related product areas Application
Nunclon sphera 3D culture systems Spheroid growth plates
Cell tracing, tracking, and morphology dyes T cell labeling for imaging
Imaging cell proliferation dyes Cell proliferation dyes for high content analysis
EVOS imaging systems EVOS imaging systems allow you to image and analyze 3D cell models.
High-content screening instruments Identify the fluorescence properties of single 3D clusters within a culture of clusters in multiwell plate formats.

Additional resources for 3D cell models

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Illustration of cell model testing

Evaluating model systems for testing the efficacy of adoptive cell therapy approaches

There have been tremendous advances in mouse models and their use in determining efficacy of adoptive cell therapy products.[6–8] A variety of mouse models, including syngeneic, transgenic, xenograft, and humanized are often used to evaluate the effectiveness and potential risk of CARs. The latest humanized mouse models are better at mimicking the response from a human’s immune system.[6–8] Of course this is still not the perfect model for determining the true effectiveness in a downstream clinical setting, but closer, nevertheless. Analysis of soluble protein factors is important for understanding the mechanisms of killing and to monitor the cytokine profile that is present at multiple stages of the workflow, including downstream in vivo scenarios. The onslaught of cytokines released during CAR implantation, known as cytokine release syndrome (CRS), is responsible for off-target effects and can lead to organ failures and neurological disorders.[6–8] Therefore during CAR treatment in these models it is necessary to evaluate the presence of other cell types and their exhibited phenotypes, the cytokine profiles produced, tumor size reduction, and survival rates. By combining various approaches for protein detection these assessments can be made in the model of choice.

Learn more about the tumor microenvironment and biomarker discovery process

Related product areas Application
Antibodies Primary antibodies recognize specific cellular markers and can serve as useful tools to characterize cell lines.
Immunoassays From single analyte identification to multiplexed formats Invitrogen immunoassays offers a suite of options for biomarker identification, quantification and analysis.
Pro-Detect Rapid Assay Kit Fast and efficient evaluation of protein expression.

Additional resources for protein detection assays

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Illustration showing T cell capture by Dynabeads magnetic beads

T cell isolation

The process of CAR cell engineering and therapy starts and ends with the subject. A blood sample is taken is taken from the subject and leukocytes are separated from other non-leukocyte populations, through a process called leukapheresis.[2,3] Mononuclear cells can then be purified using density gradient centrifugation and analyzed for cell viability and expression of immune effector-cell markers prior to activation and expansion for downstream cell engineering. In one study performed by Dr. Charles Prussak, donor-purified lymphocytes were analyzed on the Attune NxT Flow Cytometer to determine the relative CD4 and CD8 T cell populations (Figure 1).

4 panel flow cytometry scatter plots showing event acquisition and gating to reveal CD4- and CD8-expressing cells
 Click to enlarge

Figure 1. Flow cytometry analysis of purified peripheral mononuclear blood cells (PBMCs). Isolated PBMCs were analyzed by the Attune NxT Flow Cytometer for viability and CD4 and CD8 T cell populations. In the first plot (upper left), forward and side scatter channels clearly demonstrate a mixture of lymphocytic and non-lymphocyte populations based on size and granularity. Gating was first performed on the lymphocyte-sized population and analyzed for cell death based on the presence of positively stained cells (top right). Further analysis for CD3 (bottom left), CD4, and CD8 expressing cells (bottom right) revealed an expected 2:1 ratio of CD4 to CD8 cells.

Immune cell activation

Immune cells within isolated lymphocyte populations require activation and expansion prior to genetic modifications. For this purpose, it is critical to mimic antigen presentation during immune cell culture. For T cell activation, magnetic beads bound to CD3 and CD8 monoclonal antibodies, combined with activating cytokines, such as IL2, IL7 or IL15, are an effective way to mimic B cell antigen presentation and to achieve activation. This can be performed with Dynabeads CD3/CD28 in Gibco CTS OpTmizer T Cell Expansion SFM. Following incubation, as seen in Figure 2, the population then can be analyzed for proliferation and activation markers such as HLA DR, CD22, or CD69. This is an important step prior to transduction and genetic modification of cells, as over activation can cause activation-induced death. Additional analysis can be performed for cell size, viability, and proliferation, as shown in Figure 3, with flow cytometry or microscopy analysis. Indicators of health and cell function during expansion are important factors to evaluate for many downstream immunological research application areas.

4 panel flow cytometry histogram showing an increase in the number of HLA DR-expressing cells
 Click to enlarge

Figure 2. Flow cytometry analysis of activated T cells prior to transduction. Incubation and activation were performed over a 72 hr time course using magnetic CD3/CD28 activation beads. Following activation, analysis was performed on CD4- and CD8-positive T cells using Attune NxT Flow Cytometer. There is increased expression of lymphocyte populations based on activation associated antigen HLA DR compared to the 72 hr population (bottom row) with the initial time point of 0 hours (top row).

Flow cytometry histogram of the number of cells as a function of cell size in activated and non-activated T cells
 Click to enlarge

Figure 3. Quantification of T cell viability and concentration after activation. Isolated human T cells were activated using Dynabeads CD3/CD28 Untouched Human T Cell Kit in Gibco CTS OpTmizer T Cell Expansion SFM. Cells were stained with Invitrogen Trypan Blue Stain (0.4%) prior to analysis on the Countess II FL Automated Cell Counter. Beads within the sample can be gated out based on size so that only the T cell population is part of the downstream analysis. As expected, activated T cell populations appeared greater in size than non-activated T cells.

Related product areas Application
T cell product selection guide Find culturing and activation products for cell therapy
Growth factors for cell therapy products Find the appropriate growth factor for T cells
Automated cell counters For cell counting, viability and size determination
T cell expansion in clinical research Isolate, activate and expand T cells

Additional resources for T cell isolation and activation

Cancer research webinar

Presented by:
Charles Prussak, Pharm D, PhD, UCSD Moores Cancer Center

Title:
The Complex Pharmacology of T-Cell CARs

Broadcast dates:
Dec 12 & 13, 2017

Read the press release:
First FDA-approved cell therapy for leukemia utilizes Thermo Fisher Scientific’s CTS Dynabeads Technology

TOP

illustration showing CAR T genetic modification

CAR T cell genetic modification approaches

Classical retroviral or lentiviral transduction is typically used for CAR cell generation,[4,5] although there is an increased interest in pursuing the use of CRISPR/Cas9 gene editing procedures for adoptive cell transfer immunotherapies. New tumor-specific targets of interest identified through biomarker discovery efforts are under investigation by the research and translational community. For instance, Dr. Prussak’s lab is interested in ROR1, a protein normally found in the second trimester of fetal nervous system development is now being identified in some hematological or solid tumor cancers, making it an ideal target for immune therapy. To target this protein, viral particles containing the ROR1 antigen in the context of a CAR construct, were transduced into the activated T cells from Figure 3. Flow cytometry analysis was performed to determine viral infection efficiency by analyzing CD4- and CD8-positive T cells for ROR1 expression at the cell surface (Figure 4).

PBMC transduction results - control
 Click images to enlarge
PBMC transduction results - moi 1
PBMC transduction results - moi 3

Figure 4. Lentiviral transduction of viral particles carrying ROR1 CAR. Donor PBMCs were isolated and activated prior to transduction. ROR1 CAR–containing viral particles were used to transduce cells at multiplicities of infection (MOI) of 1 and 3. CD4- and CD8-positive cells were labeled with anti-ROR1 antibody recognizing the single chain variable fragment (scFv), and analyzed for positive fluorescence on the Attune NxT Flow Cytometer.

Related product areas Application
CRISPR/Cas9 Gene editing
Lentiviral production systems Cell engineering
Cell viability dyes Cell death assays for flow cytometry
CellTrace dyes Cell proliferation for flow cytometry

CAR T cell genetic assessment

Process quality control and evaluation during CAR development is necessary in both basic and translational settings to ensure efficacy of the engineered effector cell. Phenotypic assessment using antibody labeling and function-based approaches is needed, but genetic diversity and the clonal distribution of the T cell are variables that can lead to reduced efficacy and must also be evaluated. Next-generation sequencing (NGS) analysis with the Oncomine TCR Beta-LR Assay can be used at all stages of the CAR workflow to assess evenness, a measurement of a population’s T cell receptor (TCR) distribution within the T cell repertoire.

Related product areas Application
Oncomine TCR Beta-LR Assay Measure the TCRβ repertoire
Ion GeneStudio S5 systems Next-generation sequencing (NGS)

Additional resources for CAR T cell genetic engineering

TOP

Illustration of CAR T cell-mediated killing

CAR T cell killing assays

Researchers can study CAR T–mediated cell killing in cell co-culturing assays, with both 2D and 3D models. Sourcing the appropriate cells that mimic the disease models of interest can aid in the study of the effectiveness and specificity of a generated CAR model. The ROR1 CAR T cell line from Figure 4 was used in a mixed cell culture to validate its ability to kill a ROR1-positive B cell lymphocyte cell line called Jeko. To do so, ROR1 CAR T cells were incubated with Jeko cells labeled for identification and for tracking proliferation and were analyzed at 1 and 43 hours (Figure 5). At the 43-hour time point, gating on cells positive for CellTrace Yellow dye shows a significant reduction of Jeko cells compared to control (43% to 5.5%), indicating T cell–mediated killing of the Jeko cell line. However, it cannot be determined from this data whether or not the killing was achieved specifically through ROR1 binding to its target.

Flow cytometry scatter plots and histograms showing Jeko lymphoma cell death following exposure to ROR1-engineered CAR T cells
 Click to enlarge

Figure 5. CAR T cell killing of Jeko cells. Engineered ROR1 CAR T cells were incubated at a 1:1 ratio with Jeko B cell lymphoma cancer cell line. Jeko cells were stained with CellTrace Yellow dye prior to incubation and analyzed for expression at different time points. Analysis at 1 hour post incubation shows 45% of cells detected in the yellow channel on Attune NxT Flow Cytometer. After 43 hours, the analysis was performed again and only 5.5% of cells exhibited yellow fluorescence. Loss of fluorescence here indicates that the ROR1 CAR T cell killed Jeko cells.

Assessing engineered CAR T cell targeting

In a subsequent experiment using a similar co-culturing assay, two separate dyes were used to label Mec 1 cells that were engineered to express ROR1 as well as a population that remained un-altered. ROR1 CAR T cells were co-cultured at a ratio of 3:1:1 with ROR1-positive Mec cells and ROR1-negative Mec1 cells, respectively. As seen by flow cytometry analysis, ROR1 CAR T cells can differentially kill the target-expressing cells (Figure 6). When comparing all co-culturing experiments (Figure 5 and 6) it can be determined that this process is both dose and time dependent (Figure 7). It is important to note that if the ratio of CAR T cell to ROR1-negative Mec cells is increased to 10:1, a large percentage of Mec cells killed as an off-target effect is observed. Conversely with some non-CAR T cells there is also killing of the cells in co-culturing experiments, further demonstrating the subtilties of T cell killing and off-target effects. Off-target killing remains a problem for both autologous and allogeneic, donor-derived, CAR T cells and is a critical hurdle on the path to approval and effective administration of CAR-T cell products.[6] Ultimately, these are valuable assays to demonstrate T cell killing specificity and efficacy prior to moving into more biologically complex models.

Flow cytometry results indicate that the ROR1-expressing CAR T line is able to kill ROR1-positive Mec 1 cells
 Click to enlarge
Flow cytometry results indicate that the ROR1-expressing CAR T line is able to kill ROR1-positive Mec 1 cells

Figure 6. Co-culturing of ROR1 CAR T cells with Mec 1 leukemic cell line to show target specificity. ROR1 CAR T cell line were co-cultured at a ratio of 3:1:1 with ROR1 positive Mec1 cells and ROR1 negative Mec1 cells labeled with either Invitrogen CellTrace Red or Invitrogen CellTrace Yellow, respectively. Flow cytometry plots (top row) demonstrate gating to separate out live single cells. Initial gating was performed using forward and side scatter channels to separate background noise from cells. A viability dye was used to separate dead cells from the population, followed by single-cell gating. Finally, red and yellow channels were used to analyze the population for the relative amounts of Mec 1 positive and negative cells (bottom row). Analysis was compared at 1-hour (top group) and 20-hour (bottom group) post incubation on the Invitrogen Attune NxT Flow Cytometer.

Quantification of co-culturing experiments
 Click to enlarge

Figure 7. Quantification of co-culturing experiments from Figures 5 and 6. Each co-culturing scenario was quantitated and compared in a dose- and time-dependent manner to determine the effect of ROR1 CAR T cells. When the data is normalized there is an increased reduction of JeKo cells in the presence of ROR1-CAR T cells in a time- and dose-dependent manner. Similarly, there is large reduction of ROR1-positive Mec1 cells labeled with CellTrace Red, when compared to ROR1 negative Mec1 cells. This indicates ROR1-targeted CAR T cells are able to target and specifically kill ROR1-expressing Mec 1 cells in a time- and dose-dependent manner.

Related product areas Application
Cell viability dyes Cell death assays for flow cytometry
CellTrace dyes Cell proliferation for flow cytometry
Antibodies Primary antibodies recognize specific cellular markers and can serve as useful tools to characterize cell lines.

Additional resources for functional assays for CAR T cells

TOP

Illustration of CAR T cell-mediated killing

Assays for T cell killing using 3D models

3D models in the context of cancer (e.g., tumor spheroids or tumoroids) have demonstrated more biological relevancy than traditional 2D cell cultures, as they provide a context that more closely resembles the tumor microenvironment. 3D tumor models are being used more frequently in combination with adoptive cell therapy approaches. Researchers are better able to understand effector cell attributes like invasion, potency, oxidative stress, and apoptosis in the context of 3D tumor models. For instance, Figure 8 represents T cell killing through apoptosis of lung cancer spheroids. In this experiment, T cells are labeled with a far-red–fluorescent cell tracer, and spher¬oids are labeled with an apoptosis indicator. As the T cell dose increases, an increasing number of A549 cells in a lung cancer spheroid undergo apoptosis, which was quantified using the mean signal intensities of cells within the spheroid.

Figure 8. T cell–dependent killing of lung cancer spheroids. T cells were isolated from human peripheral blood and expanded for 5 days with Gibco Dynabeads Human T-Activator CD3/CD28 in Gibco CTS OpTmizer T Cell Expansion SFM. Cells were analyzed with the Invitrogen Countess II FL Automated Cell Counter to determine cell viability and concentration, and cell concentration was adjusted to 1 x 106 cells/mL in Gibco PBS, pH 7.2. T cells were then labeled with 2 μM Invitrogen CellTracker Deep Red Dye (purple) for 15 min and washed with CTS OpTmizer medium. For spheroid formation, A549 (adenocarcinomic human alveolar basal epithelial) cells were plated in a Thermo Scientific Nunclon Sphera 96-well microplate at a density of 7,500 cells/well, incubated overnight in a cell culture incubator with 5% CO2 at 37°C, and analyzed on the Invitrogen EVOS XL Core Imaging System to confirm spheroid formation. Invitrogen CellEvent Caspase-3/7 Green Detection Reagent (2 μM final concentration) (green) and the indicated number of labeled T cells were added to each well, and the mixture was incubated for 4 hr at 37°C. (A) Two examples of the response of a lung cancer spheroid to 4 different T cell concentrations are shown, each imaged on the Thermo Scientific CellInsight CX7 LZR HCA Platform using confocal mode with 10 μm Z slicing. (B) Apoptosis in A549 spheroids and the T cell dose response were quantified using the CellInsight CX7 LZR platform and Thermo Scientific HCS Studio Cell Analysis Software. The spheroids were segmented as single objects based on the brightfield image using HCS Studio software, and cells were counted within the object. Mean signal intensities of cells within the spheroid were used to quantify immune cell cytotoxicity.

If available, these models can provide a relevant context for subject-specific responses and can contribute to advances in the field of personalized cancer immunotherapy. CAR T cells engineered with a subject-specific tumor antigen receptor molecule and then co-cultured with cancer spheroids can provide a context for assessment of tumor invasion and killing by the T cell. As proof of concept, Figure 9 demonstrates that by increasing the ratio of CAR T cells to tumor spheroids there is a clear destruction and lysing of the cultured spheroid. This experiment combines a broad range of products that enables an elegant demonstration of visualizing cell-to-cell interactions between engineered immune cells and tumor spheroids.

Fluorescence microscopy images of cell lysis in cancer spheroids following exposure to CAR T cells

Figure 9. Chimeric antigen receptor (CAR) T cell invasion into cancer spheroids. HCC827 spheroids were formed using spheroid microplates for 48 hr. Then, 24 hr after the addition of EGFR scFv-CD28-CD3ε CAR T cells (ProMab Biotechnologies), spheroids were immunostained for cytokeratin-7 (green) and CD3ε (red), and counterstained with Hoechst dye (blue). As the effector-to-target ratio is increased from 10:1 (middle panel) to 40:1 (right panel), invasion of the CAR T cells into the HCC827 tumor spheroid and subsequent tumor cell lysis are visible. Images were obtained on the Thermo Scientific CellInsight CX7 High-Content Analysis Platform in confocal mode with a 10x objective, and used with permission from Corning Inc.

Related product areas Application
Nunclon sphera 3D culture systems Spheroid growth plates
Cell tracing, tracking, and morphology dyes T cell labeling for imaging
Imaging cell proliferation dyes Cell proliferation dyes for high content analysis
EVOS imaging systems EVOS imaging systems allow you to image and analyze 3D cell models.
High-content screening instruments Identify the fluorescence properties of single 3D clusters within a culture of clusters in multiwell plate formats.

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Illustration of cell model testing

Evaluating model systems for testing the efficacy of adoptive cell therapy approaches

There have been tremendous advances in mouse models and their use in determining efficacy of adoptive cell therapy products.[6–8] A variety of mouse models, including syngeneic, transgenic, xenograft, and humanized are often used to evaluate the effectiveness and potential risk of CARs. The latest humanized mouse models are better at mimicking the response from a human’s immune system.[6–8] Of course this is still not the perfect model for determining the true effectiveness in a downstream clinical setting, but closer, nevertheless. Analysis of soluble protein factors is important for understanding the mechanisms of killing and to monitor the cytokine profile that is present at multiple stages of the workflow, including downstream in vivo scenarios. The onslaught of cytokines released during CAR implantation, known as cytokine release syndrome (CRS), is responsible for off-target effects and can lead to organ failures and neurological disorders.[6–8] Therefore during CAR treatment in these models it is necessary to evaluate the presence of other cell types and their exhibited phenotypes, the cytokine profiles produced, tumor size reduction, and survival rates. By combining various approaches for protein detection these assessments can be made in the model of choice.

Learn more about the tumor microenvironment and biomarker discovery process

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Antibodies Primary antibodies recognize specific cellular markers and can serve as useful tools to characterize cell lines.
Immunoassays From single analyte identification to multiplexed formats Invitrogen immunoassays offers a suite of options for biomarker identification, quantification and analysis.
Pro-Detect Rapid Assay Kit Fast and efficient evaluation of protein expression.

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Citations

  1. Rosenberg S, et al. (1998) Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. Preliminary report, N Engl J Med 319:1676–1680.
  2. Rosenberg S, et al. (2008) Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 8:299–308.
  3. Zhang C, et al. (2017) Engineering CAR-T cells. Biomark Res 5:22.
  4. Brocker T, et al. (2000) Chimeric Fv-zeta or Fv-epsilon receptors are not sufficient to induce activation or cytokine production in peripheral T cells. Blood 96:1999–2001.
  5. Salmikangas P, et al. (2018) Chimeric antigen receptor T-cells (CAR T-cells) for cancer immunotherapy – moving target for industry? Pharm Res 35:152.
  6. Wang P, et al. (2018) Preclinical models in chimeric antigen receptor-engineered T-cell therapy. Review Article, Human Gene Therapy 29:534–546.
  7. Yu X, et al. (2018) Modelling CART therapy in humanized mice. Commentary. EBioMedicine 40:25–26.
  8. Jin C H, et al. (2019) Modeling anti-CD19 T cell therapy in humanized mice with human immunity autologous leukemia. EBioMedicine 39:173–181.

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