Chimeric antigen receptor cell therapy (CAR-T)

Chimeric antigen receptor (CAR)-T cell therapy has attracted a significant amount of attention as a result of its recent clinical success in B-cell malignancies. Generally, the CAR-T cell discovery process is comprised of CAR identification, T-cell activation, transduction, and expansion, as well as assessment of CAR-T cytotoxicity.

The evaluation methods currently in use for the CAR-T discovery process can be labor-intensive, with low-throughput while necessitating the preparation of numerous sacrificial samples to generate kinetic data.

Throughout this study, the use of a plate-based image cytometer was employed to monitor anti-CAIX (carbonic anhydrase IX) G36 CAR-T production and evaluate its cytotoxic potency of direct and selective killing against CAIX+ SKRC-59 human renal cell carcinoma cells.

The transduction efficiency and cytotoxicity results were evaluated using image cytometry and directly compared to flow cytometry and Chromium 51 (51Cr) release assays, demonstrating that image cytometry was comparable against these traditional methods.

The image cytometry method simplifies the required assays throughout the CAR-T cell discovery process by evaluating a plate of T cells from CAR-T production to in vitro functional assays with minimal disruption.

The proposed method can limit assay time and uses fewer cell samples by imaging and analyzing the same plate over time without the need to compromise any cells. The capacity to monitor kinetic data can facilitate further insights into the behavior and interaction between CAR-T and target tumor cells.

Key terms

CAR-T discovery; cytotoxicity assessment; label-free cell proliferation; transduction efficiency; chromium release; flow cytometry; image cytometry

Chimeric antigen receptor (CAR)-T cell therapy is dramatically transforming the field of cancer immunotherapy.

T cells are engineered to express CAR, which is comprised of a single-chain variable antibody fragment (scFv) paired with an intracellular signaling motif that includes CD3ζ (z) activation domain (first generation), with CD28 or 41BB co-stimulatory domain (second generation), or both (third generation).1

When binding tumor antigen to CAR, the intracellular signaling domain provokes T cell activation, leading to CAR-T proliferation, cytokine release, and tumor cells killing in an antigen-dependent manner.

Since the treatment of antiCD19 CAR-T cell therapy in B cell malignancies was first reported in 2010,2,3 the field has increased considerably and immuno-oncology researchers are working to apply this cellular therapy to various types of cancers including solid tumors.

The recent FDA approval of Kymriah (tisagenlecleucel) and Yescarta (axicabtagene ciloleucel) has introduced this new type of cellular immunotherapy into mainstream cancer therapy which has contributed further to the increase in the development of CAR-T cells with improved efficacy and safety profiles.4

The CAR-T discovery process is made up of multiple parts: identification of CAR candidates that can precisely target tumor-associated antigens (TAAs) that are overexpressed on cancer cells, transient transfection or stable transduction of activated T cells to provoke CAR expression, followed by CAR-T cell expansion, as well as efficacy and safety assessment.5,6

Presently, Kymriah and Yescarta as well as several clinical trials CAR-T cells, are created by lentiviral or gamma-retroviral transduction. Numerous technologies and detection methods are used to acquire the range of stepwise evaluations that are necessary throughout the CAR-T discovery process.

However, these methods can be complicated when screening the CAR candidates in a high-throughput manner.7 CAR-T cell transduction efficiencies are generally measured using a flow cytometer,8,9 which can take up to 2 minutes per sample; this adds up to 2–3 hours per plate, including warm-up and cleaning procedures.

Therefore, for high-throughput, flow cytometry is not a suitable method as measurements cannot be conducted in a timely manner. Furthermore, it is not possible to reuse the precious CAR-T cell samples used for flow cytometry for downstream assays.

Finally, CAR-T potency is typically measured using 51Cr release assay, which is a radiological hazard, requiring a considerable amount of target and effector cells and indirectly measures only cell death via the supernatant.10-12

In the last decade, image cytometry technologies have made major improvements from single sample measurement to high-throughput plate-based analysis. Previously, image cytometry has shown improvements to cell-based assays, such as transduction efficiency,13,14 cell proliferation15-17, and cell killing assay.18-21

Generally, the system can image and analyze a complete 96-well plate utilizing bright field and fluorescence in less than 5 and 10 minutes, respectively. Thus, the time required to assay transduction efficiency, cell proliferation, and cell killing can be reduced considerably.

This process necessitates approximately 20 times fewer effector cells, which is extremely beneficial when working with valuable samples. Since image cytometry can measure cells in plates directly, the cells are not disturbed and can be tracked over multiple time points.

Moreover, cell images are saved and viewed, and the analysis can therefore be validated to eradicate any uncertainties.

This article will seek to demonstrate that the use of image cytometry is crucial when conducting cell-based assays for CAR-T discovery and streamlining the process.

To start, CAR-T cell transduction efficiencies and proliferation were measured utilizing image cytometry, where the transduction efficiencies were directly compared to flow cytometry for verification.

Subsequently, a direct tumor cell killing assay was performed with the anti-CAIX G36-CD28 CAR-T cells, previously reported, 22 on CAIXexpressing human clear cell renal cell carcinoma (ccRCC) SKRC-59 cells using image cytometry and compared to 51Cr release.

Finally, a selective tumor cell killing assay was carried out with the anti-CAIX G36-CD28 CAR-T cells and a co-culture of CAIX-positive and CAIX-negative SKRC-59 cells using image cytometry for comparison with flow cytometry.

The results of each experiment were comparable between image cytometry and the relevant traditional methods, which endorsed the use of image cytometry for CAR-T discovery assays.

In addition, the benefits brought forth by image cytometry technologies have been used to enhance the efficiency of data collection, produce time-course data, limit the time taken for each assay, boost consistency and reduce sample waste.

The proposed image cytometry method can deliver a streamlined CAR-T discovery process, in which, with minimal disruption, and direct analysis of a plate of CAR-T cells from generation to in vitro functional assays.

Materials and methods

Production of lentivirus particles

HEK 293T (CRL-11268, ATCC) and Lenti-X 293 T (Clontech Laboratories, Mountain View, CA) cells were cultivated in DMEM medium (Life Technologies, Carlsbad, CA) with an addition of 10% FBS, 100 IU/mL penicillin, and 100 μg/ mL streptomycin.

Lentiviruses were generated by transient five plasmids co-transfection into Lenti-X 293 T cells using polyethyleneimine (PEI).23

For each individual 80% confluent Lenti-X 293 T cells in a 15 cm plate (Corning, Corning, NY), 60 μg of total five plasmids were transfected, 20 μg of the CAR encoding plasmid, 10 μg of each structural plasmid pHDH-Hgpm2 (HIV gag-pol), pMD-tat, pRC/CMV-rev and Env VSV-G. The virus supernatant was concentrated with a homemade 4X concentrator.24,25

The virus pellet was resuspended with RPMI 1640 medium (Life Technologies) and frozen at −80 ℃. The G36-CD28 (targeting CAIX) and A716-CD28 (targeting B-cell maturation antigen, BCMA) lentiviruses for CAR-T have been previously generated.

Finally, the blue fluorescent protein (BFP) and mCardinal lentiviruses were produced in a similar fashion.22 Lentiviruses titration was conducted using HEK 293 T cells. The 1 × 105 293 T cells were seeded in 24-well plates and transduced the next day with various folds of diluted lentiviruses.

Forty-eight hours post-transduction, transduction efficiency was evaluated using flow cytometry and the multiplicity of infection (MOI) was established for each lentivirus.

Preparation of human renal carcinoma target cells

Dr. Gerd Ritter from Memorial Sloan-Kettering (New York, NY) generously donated human renal cell carcinoma (SKRC-59) cells; the cells were mycoplasma negative tested in lab using MycoAlert mycoplasma detection kit (Lonza).

The cells were cultured at 37 ℃ at 5% CO2 in RPMI with an addition of 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco, Carlsbad, CA), 100 IU/mL penicillin and 100 μg/mL streptomycin.

SKRC-59 cells were manipulated to become CAIX+ SKRC59 and CAIX SKRC-59 as previously described by lentivirus transduction22,26 or CRISPR/Cas9 as the wild-type cells are CAIX negative.

Moreover, mCardinal and BFP lentiviruses were employed to transduce the CAIX+ SKRC-59 and CAIX SKRC-59 cells to express nuclear mCardinal and BFP fluorescence (CAIX+ mCardinal+, CAIX BFP+ ).

Preparation of CD8+ T cells

Leukocyte reduction system (LRS) cones gathered from healthy donors were acquired from the blood bank of the Brigham and Woman’s Hospital (Boston, MA) under a Dana Farber Cancer Institute approved human protocol 14-343.

Human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque PLUS (GE Healthcare, Chicago, IL). The microbeads for CD8 positive selection (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to separate CD8+ T cells from PBMCs.

CD8+ T cells were then excited with TransAct CD3/CD28 nanobeads (Miltenyi Biotec). CD8+ T cells were retained in complete RPMI media comprised of an additional 20 mM HEPES, and every other day 50 IU/mL of IL-21 (Miltenyi Biotec) was added to the medium.

CAR-T cell transduction analysis and proliferation assay using image cytometry

Around 5 × 105 of CD8+ T cells were fed into each well of a 6-well plate (Corning Costar) with 50 IU/mL of IL21 and TransAct activation beads. Twenty-four hours after activation, the cells were transduced with the lentiviruses at an MOI of 20 and 10 μg/mL DEAE, producing ZsGreen fluorescence.

The T cells were centrifuged at 500g for 2 minutes and then counted using the image cytometer. Twenty hours after CAR lentivirus transduction, the transduced T cells were washed and held in fresh culture media.

Later, the transduced T cells were imaged and analyzed in bright field and green fluorescence channels at intervals of 24 and 48 hours after the transduction. The transduction efficiency was determined by the equation:

Transduction efficiency

For proliferation monitoring, T cells were centrifuged at 500g for 2 minutes and counted directly in bright-field images with the application of image cytometry at 0, 24, and 96 hours of incubation to establish T-cell proliferation. Each cell type (G36-CD28, A716-CD28, and untransduced T cells) was conducted at n = 6.

CAR-T cell transduction analysis using flow cytometry

CAR-T generation is detailed above. The CAR-T transduction efficiency was characterized (n = 6) by measuring the green fluorescent population (ZsGreen+) with an LSR Fortessa (BD Biosciences, San Jose, CA).

CAR-T cell killing assay using chromium 51 release

Preparation of an SKRC-59 target cell sample was performed at 1.25 × 107 cells/mL (1 × 106 cells in 80 μL of T cell media). Subsequently, 25 μL of 2 mCi/mL 51Cr, Perkin Elmer NEZ030002M) were introduced to the target cells and incubated at 37 ℃ for 1 hour.

Throughout the incubation process, preparation of the effector cells involved adding 50 μL into a V-bottom 96-well plate (Corning Costar). More control wells were assembled with target cells only (spontaneous release) and target cells with 1% Triton-X (maximum release).

Once the 51Cr labeling was completed, the target cells were washed two to three times using the RPMI complete medium.

The labeled cells were then resuspended in RPMI complete medium (RPMI supplemented with 10% (v/v) heat-inactivated fetal bovine serum; FBS, Gibco, Carlsbad, CA) to generate 2,500 cells per 50 μL (5 × 104 cells/mL). About 12,500 CAR-T cells (50 μL) were introduced into the V-bottom 96-well plate and pipetted to mix the effector and target cells with E:T ratio of 5:1.

The plate was then centrifuged for 5 minutes at 1000 RPM and incubated for 4 hours at 37 ℃. Next, 100 μL of scintillation fluid was introduced to a counting plate (clear 96-well flexible PET microplate with a round bottom; PerkinElmer, Waltham, MA).

Subsequently, the experimental plate was centrifuged at 1400 RPM for 5 minutes, and 30 μL of the supernatant was moved to the counting plate and mixed using a pipette. The counting plate was read immediately in the scintillation counter. The Specific Lysis was determined using the following equation:

Specific Lysis

CAR-T cell killing assay of a single target using image cytometry

Twenty milliliters of CAIX+ mCardinal+ SKRC-59 target cells at a concentration of 1.5 × 104 cells/mL were acquired to prepare 3 × 105 total target cell count. Around 3,000 target cells were fed in a 96-well plate (Greiner 655,090) in 200 μL/well.

After 12 hours of incubation, the plate was scanned and analyzed in bright field and far-red channel for mCardinal as the zero-hour time point. Next, CAR-T cells were introduced at various E:T ratios (1:1, 5:1, and 10:1). Correction of the number of cells introduced to the experiment was completed by the transduction efficiency percentage.

Additional control wells were assembled with target cells only (negative control) and target cells with 1% Triton-X (positive control). Next, the plate was scanned and analyzed at 24- and 48-hour time-points with the following equation:

Inhibition

Each cell type (G36-CD28, A716-CD28, and untransduced T cells) and E:T ratio was performed in triplicate.

Selective CAR-T cell killing assay using flow cytometry

A combination of 6 × 105 CAIX+ BFP+ and 6 × 105 CAIX BFP+ SKRC-59 target cells were prepared to a concentration of 1 × 105 target cells and 1 × 105 nontarget cells/mL together in 6 mL.

Around 1 × 105 target cells were fed with the same amount of non-target cells in a 6-well plate at 1 mL/well. After 12 hours of incubation, CAR-T cells were introduced at E:T ratio of 10:1.

Additional control wells were assembled with target + non-target cells only and the target + non-target cells and untransduced T cells (negative control) mixture. After 24 hours of co-culture, all of the cells from each well were gathered together and stained with PE-conjugated anti-human CAIX antibody (Miltenyi Biotec, 130-110-057).

All samples were evaluated using an LSR Fortessa (BD Bioscience), and data analysis was performed using FlowJo software.

Selective CAR-T cell killing assay using image cytometry

Preparation of a mixture of 3 × 105 CAIX+ mCardinal+ and 3 × 105 CAIX BFP+ SKRC-59 target cells was completed to a concentration of 1.5 × 104 target cells +1.5 × 104 nontarget cells/mL in 20 mL.

On average, around 3,000 target cells were fed with the same amount of non-target cells in a 96-well plate at 200 μL/well (Greiner, 655090).

After 12 hours of incubation, the plate was scanned and analyzed in bright field, far-red channel for mCardinal, and blue channel for BFP as the zero-hour time point. Subsequently, CAR-T cells were added at E:T ratio of 2:1 and 10:1.

Extra control wells were assembled using mixed cancer cells only (negative control) and cells with 1% Triton-X (positive control). Next, the plate was scanned and analyzed at 24 and 48 hour time points.

The selective killing index was determined using the following equation:

The selective killing index

Each cell type (G36-CD28, A716-CD28, and untransduced T cells) and E:T ratio was performed in triplicate.

Image cytometry system for CAR-T development

The Celigo Image Cytometer (Nexcelom Bioscience, Lawrence, MA) was used for conducting CAR-T cell-based assays.

It utilizes one bright field (BF) and four fluorescent (FL) imaging channels, blue (EX377/50 nm, EM470/22 nm), green (EX 483/32 nm, EM 536/40 nm), red (EX 531/40 nm, EM 629/53 nm) and far red (EX 628/40 nm, EM 688/31 nm) for high-throughput cell-based assays (27-30).

Cells planted in traditional 96-well microplates were autofocused, scanned, and analyzed in the software, which comprised of five major steps: START, SCAN, ANALYZE, GATE and RESULTS.

The software Application “Target 1 + 2” was used for determining transduction efficiency by counting total CAR-T cells in the bright field channel (exposure: auto) and ZsGreen+ (exposure: 150 ms) cells in the green channel to calculate the ZsGreen percentages.

The ANALYZE parameters for the green channel were fixed to: “Algorithm = Fluorescence,” “Intensity Threshold = 2,” “Precision = High,” “Cell Diameter = 20,” “Dilation Radius = 0,” “Background Correction = Check,” “Separate Touching Objects = Check,” “Minimum Cell Area = 35.”

The ANALYZE parameters for the bright-field channel were set to: “Algorithm = Fluorescence,” “Intensity Threshold = 4,” “Precision = High,” “Cell Diameter = 10,” “Dilation Radius = 0,” “Background Correction = Uncheck,” “Separate Touching Objects = Check,” “Minimum Cell Area = 35.”

For cell proliferation assay, the “Direct Cell Counting” Application was employed for direct counting of CAR-T cells in bright field channel (exposure: auto) in each well over time.

The ANALYZE parameters for the bright-field channel were fixed at: “Algorithm = Fluorescence,” “Intensity Threshold = 4,” “Precision = High,” “Cell Diameter = 10,” “Dilation Radius = 0,” “Background Correction = Uncheck,” “Separate Touching Objects = Check,” “Minimum Cell Area = 35.”

The “Target 1 + 2” Application was used for counting the number of fluorescent positive target cells in blue or far red channels for the single target cell killing assay (mCardinal or BFP only).

Finally, the “Target 1 + 2 + 3” Application was employed for selective CAR-T killing of BFP+ or mCardinal+ target cells, which used bright field, blue, and far-red channels. The image cytometer was employed to directly count the number of mCardinal and BFP positive cells in the wells at various effector: target (E:T) ratios over time.

The results of the counting process were used to determine the level of CAR-T cell-mediated cytotoxicity. The exposure time and gain setting for blue and far-red channels were set to 120 ms and 200, respectively.

The ANALYZE parameters for the blue channel were fixed at: “Algorithm = Fluorescence,” “Intensity Threshold = 2,” “Precision = High,” “Cell Diameter = 20,” “Dilation Radius = 0,” “Background Correction = Uncheck,” “Separate Touching Objects = Uncheck,” “Minimum Cell Area = 10.”

The ANALYZE parameters for the far red channel were set to: “Algorithm = Fluorescence,” “Intensity Threshold = 2,” “Precision = High,” “Cell Diameter = 20,” “Dilation Radius = 0,” “Background Correction = Uncheck,” “Separate Touching Objects = Uncheck,” “Minimum Cell Area = 100.”

Results

CAR-T transduction efficiency and proliferation measurement

Flow cytometric analysis is the traditional method used to evaluate transduction efficiency; however, it produces a significant amount of cell waste because the samples used in flow cytometry cannot be reused for downstream assays.

To save CAR-T samples and effectively track the production of CAR-T cells, the development of an image cytometry method was carried out to measure the CAR-T transduction efficiency and track cell proliferation.

CD8+ T cells were separated from healthy donor blood utilizing CD8 microbeads and then seeded and activated by using anti-CD3/CD28 monoclonal antibody (mAb)-coated beads in 6-well plates on Day 0.

Lentiviruses were introduced to transduce T cells with the various CARs, anti-CAIX G36-CD28, and anti-BCMA A716-CD28 on Day 1. The images of corresponding wells were acquired at various time points; Days 0, 1, and 4 (Fig. 1a).

The cell number of each well was counted to produce the cell proliferation histograms (Fig. 1b). The G36-CD28 and A716-CD28 CAR-T cells expanded less in comparison to the untransduced T cells, which may be a result of the toxicity caused by the high transduction MOI.

After 4 days in culture, G36-CD28 and A716-CD28 expanded 15.8- and 15.4-fold, whereas untransduced T cells expanded 26.0-fold (Fig. 1b). Transduction efficiency was determined by utilizing ZsGreen+ cells divided by total cells in brightfield (Fig. 1c).

The transduction efficiencies of G36-CD28 and A716-CD28 CAR were 39.4% and 41.9%, respectively, on Day 4. The cells were also evaluated for transduction efficiency by flow cytometry with 39.7% and 43.7%.

The comparison results are displayed in Figure 1d, showing that image cytometry performed similarly to the conventional flow cytometry without wasting any samples.

T-cell transduction and proliferation results. (a) The time-dependent fluorescent images are shown on the left panel for G36-CD28 and A716-CD28 CAR-T, as well as the untransduced T cells. The ZsGreen fluorescence is clearly observed for both G36 and A716 CAR-T on Day 4. (b) T-cell proliferation results over a 4-day culture directly counted in bright field images (n = 3). (c)  Timedependent transduction efficiencies analyzed by image cytometry (n = 3). (d) Transduction efficiency results compared between image and flow cytometry (n = 3).

Figure 1. T-cell transduction and proliferation results. (a) The time-dependent fluorescent images are shown on the left panel for G36-CD28 and A716-CD28 CAR-T, as well as the untransduced T cells. The ZsGreen fluorescence is clearly observed for both G36 and A716 CAR-T on Day 4. (b) T-cell proliferation results over a 4-day culture directly counted in brightfield images (n = 3). (c)  Time-dependent transduction efficiencies were analyzed by image cytometry (n = 3). (d) Transduction efficiency results were compared between image and flow cytometry (n = 3). Image Credit: Nexcelom Bioscience LLC

Direct CAR-T cell killing assay using image cytometry

The mCardinal fluorescence was introduced into CAIX+ SKRC-59 cells by lentiviral transduction for imaging analysis by using the far-red channel. The mCardinal+ CAIX+ SKRC-59 cells were seeded into a 96-well plate on Day −1. Images were acquired on Day 0 and CAR-T cells were added to the plate using the E:T ratios 1:1, 5:1, and 10:1.

The plate was analyzed and the number of mCardinal+ cells was counted after 24 and 48 hours of incubation (Supporting Information Figure S1a,b, Figure 2a,b, Supporting Information Figure S2a,b for E:T ratio of 1:1, 5:1 and 10:1, respectively).

The inhibition of growth induced by each treatment was calculated by using the equation as follows:

The inhibition of growth

In Figure 2a,b, under the E:T ratio of 5:1, anti-CAIX G36-CD28 inhibited CAIX+ SKRC-59 cell proliferation circa 30.5% after 24 hours and 49.9% after 48 hours of incubation, while irrelevant antiBCMA A716-CD28 CAR-T cells did not show any inhibitory activities (Fig. 2c).

This result was comparable to the 39.3% lysis obtained from 4 hour Chromium 51 release assay in the presence of G36-CD28 CAR-T cells (Fig. 2d).

The inhibition results for 1:1 and 10:1 ratios are shown in the Supporting Information Figures S1c and S2c, where the 1:1 ratio showed no inhibition effects, and the 10:1 showed higher inhibition values compared to the 5:1 with 39.1% and 65.5% at 24 and 48 hour time points.

Direct CAR-T cell-mediated cytotoxicity results for E:T ratio of 5:1. (a) The time-dependent fluorescent images are shown on the left panel for mCardinal+ CAIX+ SKRC-59 cells treated with G36-CD28 and A716-CD28 CAR-T, as well as the untransduced T cells. (b) Direct live tumor cell counting results over a 2-day co-culture measured in the far red channel by image cytometry (n = 3). (c) Timedependent tumor cell growth inhibition percentages analyzed by image cytometry. (d) Lysis percentages measured by chromium 51 release assay.

Figure 2. Direct CAR-T cell-mediated cytotoxicity results for E:T ratio of 5:1. (a) The time-dependent fluorescent images are shown on the left panel for mCardinal+ CAIX+ SKRC-59 cells treated with G36-CD28 and A716-CD28 CAR-T, as well as the untransduced T cells. (b) Direct live tumor cell counting results over a 2-day co-culture measured in the far red channel by image cytometry (n = 3). (c) Time-dependent tumor cell growth inhibition percentages were analyzed by image cytometry. (d) Lysis percentages measured by chromium 51 release assay. Image Credit: Nexcelom Bioscience LLC

CAR-T cell-selective killing assay using image cytometry

The mCardinal was introduced into CAIX+ SKRC-59 cells and BFP was introduced into CAIX SKRC-59 cells by lentiviral transduction for imaging analysis. The mCardinal+ CAIX+ SKRC-59 cells were mixed 1:1 with BFP+ CAIXSKRC-59 cells and both cells were seeded into a 96-well plate on Day −1.

Images were acquired on Day 0 and CAR-T cells were added to the plate at E:T ratios of 2:1 and 10:1. The plate was analyzed and the number of mCardinal+ and BFP+ cells were counted after 24- and 48-hour incubation (Supporting Information Figure S3a,b and Figure 3a,b for E:T ratios of 2:1 and 10:1 correspondingly).

The selectivity index was calculated using the equation as follows:

The selectivity index

For the 10:1 ratio, the G36-CD28 CAR-T cells showed a selective killing against mCardinal+ CAIX+ SKRC-59 cells over BFP+ CAIX SKRC-59 cells with a selectivity index of 2.5 after 24 hours of incubation and 4.1 after 48 hours (Fig. 3b).

These results were comparable to the results obtained from flow cytometry assays, where the selectivity index of 2.9 was obtained with 74.4% CAIX BFP+ and 25.6% CAIX+ BFP+ SKRC-59 cells (Figure 3c,d, flow cytometry gating strategies shown in the Supporting Information Figure S5).

Untransduced T cells were used as control which showed no selective killing after 24 hours co-incubation with the mixture of CAIX BFP+ and CAIX+ BFP+ SKRC-59 cells (Supporting Information Figure S4).

Selective CAR-T cell-mediated cytotoxicity results for E:T ratio of 10:1. (a) The time-dependent fluorescent images are shown on the left panel for the mixture of mCardinal CAIX+ and BFP CAIX− SKRC-59 cells treated with G36-CD28 and A716-CD28 CAR-T, as well as the Untransduced T cells. (b) Selectivity index results over a 2-day co-culture calculated with CAIX+ (far red channel) and CAIX− (blue channel) tumor cell counts measured by image cytometry (n = 3). (c) Selective killing results over a 1-day co-culture with CAIX+ and CAIX− tumor cell counts analyzed by flow cytometry. (d) Selectivity index results compared between image and flow cytometry.

Figure 3. Selective CAR-T cell-mediated cytotoxicity results for E:T ratio of 10:1. (a) The time-dependent fluorescent images are shown on the left panel for the mixture of mCardinal CAIX+ and BFP CAIX SKRC-59 cells treated with G36-CD28 and A716-CD28 CAR-T, as well as the Untransduced T cells. (b) Selectivity index results over a 2-day co-culture calculated with CAIX+ (far-red channel) and CAIX (blue channel) tumor cell counts measured by image cytometry (n = 3). (c) Selective killing results over a 1-day co-culture with CAIX+ and CAIX tumor cell counts analyzed by flow cytometry. (d) Selectivity index results compared between image and flow cytometry. Image Credit: Nexcelom Bioscience LLC

Discussion

In this study, we were able to streamline the CAR-T discovery by using the proposed high-throughput image-based detection method. Compared to the conventional methods, image cytometry was performed in a high-throughput manner with little sample waste and enabled the monitoring of CAR-T transduction, expansion, and the assessment of CAR-T killing capacity over the course of time.

It is important to note that the amount of cells required for flow cytometry in comparison to image cytometry is approximately six times more for the measurement of three time points. Furthermore, the samples used in flow cytometry cannot be used in downstream assays.

Cell expansion monitoring can also be performed using label-free or fluorescence labeling for other cell types and simultaneously assess the transduction efficiencies. An image-based cytotoxicity assay could assess target cell killing caused by chemicals, antibodies, or other reagents.

For the CAR-T killing assay, we were able to measure the direct killing of tumor cells using both Chromium 51 release and image cytometry assays. Chromium 51 release assay measured the released isotopes due to cell cytolysis and image cytometry measured the fluorescence expressed by live cells.

For the Chromium 51 release assay, when the target cell membrane is ruptured, the released isotopes are measured to determine the cytotoxicity induced by the effector cells. In contrast, the fluorescent proteins that represented live target cells required degradation time.

Due to the two different methodologies, the level of cytotoxicity was assessed at 4-hours (Chromium 51 release) and 24-hours (image cytometry) between the two methods to accurately evaluate the cytotoxicity of effector CAR-T cells.

For selective killing, we were able to assess the selectivity indices on a mixture of two different cell populations, which can also be expanded to four populations with fluorescence in green, blue, red, and far-red.

As a “living drug,” CAR-T cell has a complex preparation process. Thus, quality control (QC) is also a vital link of the streamline, which was not included in this methodology development.

For clinical development of CAR-T cells, other methods can be employed, such as the utilization of available long-term live-cell tracer and viability dyes to monitor and measure live and dead target cells over time,21 and the surface markers including CAR can be quantified by using anti-surface maker proteins or antibodies.

For example, anti-CAIX CAR transduction efficiency can be also determined by using CAIX protein conjugated with fluorophores, like DyLight amine-reactive dye (ThermoFisher), which will not require the transgene encoding fluorescent protein.

This method can eliminate the need to introduce a genetic modification to the primary cells, which is more flexible for the diversity of the patient samples and GMP quality cell therapy products. However, the diverse patient samples can also introduce variability in staining. Thus a robust and consistent method for developing CAR-T for primary tumor cells is needed.

It is important to note that the anti-CAIX antibody was employed to determine the CAIX+ and CAIX- cell populations after anti-CAIX CAR-T cell treatment to cross-validate the results obtained from image cytometry using mCardinal and BFP. In addition, the anti-CAIX antibody used for CAIX antigen staining has not observed any competition with the anti-CAIX scFvs used in the CAR construct.

There are several other conventional methods for performing high-throughput cell proliferation and cytotoxicity assays, such as calcein/LDH release, MTT-, luciferase- or cytokine secretion assays. These methods are commonly used to assess cell growth and cytotoxicity potential using a plate reader capable of detecting bright field and fluorescence light, as well as luminescence.

However, these methods are typically performed as an end-point assay, thus are unable to easily collect kinetic data.

Furthermore, the data is based on indirect measurements of the supernatant with light absorption, fluorescence, and luminescence, instead of directly counting the cells, which can be significantly affected by cell metabolic or enzymatic activities.31

There are other image cytometry systems, such as the Incucyte (Essen Bioscience, Ann Arbor, MI) and eSight (ACEA Biosciences, San Diego, CA), that can perform automated real-time image acquisition and analysis, which are widely used in research and discovery field.

CAR-T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies.32-34 However, these results have not been translatable to solid tumors due to inefficient homing of CAR-T cells, the suppressive tumor microenvironment and on-target off-tumor toxicities resulted from the sharing of CAR-T targeting epitopes on healthy tissues.35

We believe that the combination of image cytometry and other tools together will benefit CAR-T studies and accelerate the discovery process to translate this promising therapy to the treatment of solid tumors

Conclusion

The issues relating to assay time inefficiency as well as large cell numbers and their wastage during testing by conventional methods, such as flow cytometry or Chromium 51 release assay, have prompted the need to develop new detection and analysis methods for CAR-T discovery.

The advancement in imaging technologies over the last decade has allowed the development of novel image cytometers for cell-based assays, which can be applied to common assays performed during CAR-T discovery, such as cell proliferation, transduction efficiency measurement, and cytotoxicity potency evaluation.

Image cytometry provides a useful method for streamlining CAR-T discovery, from CAR-T generation to tumor-killing assessment. Compared to the conventional methods, image cytometry can reduce assay time and increase assay efficiency.

By directly analyzing and counting cells in plates without disruption, the accuracy and quality of the results can potentially be improved, as well as provide time-course monitoring of the cell samples.

Finally, the proposed image cytometry method can also be adapted to evaluate other processes, such as T-cell receptor (TCR) and antibody discovery, specifically for antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP) assays.

References

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About Nexcelom Bioscience

Nexcelom Bioscience is a developer and marketer of image cytometry products for cell analysis in life science and biomedical research. Products range from cell viability counters (Cellometer) to high throughput microwell image cytometry workstations (Celigo), used in thousands of research laboratories in academic institutes, and pharmaceutical and biotech companies. The company contributes to the life science industry through innovation and expertise in the science of cell counting.

The product family includes instruments, consumables and reagents. Nexcelom customers engage in a wide variety of research, such as cancer research, immunology, stem cell research, and neuroscience. Nexcelom offers different Cellometer models to count and analyze cell lines and primary cells, through bright field and fluorescence imaging modes. In addition, Celigo is a powerful high image quality, high throughput image cytometry system for adherent and suspension cells in microwell plates.

Nexcelom Bioscience is a fast-growing company in a huge market. With its headquarters and manufacturing facilities in the Boston area, the company currently has over 80 global employees, who are fast-paced, customer-centric, helpful to colleagues and customers, and passionate about their impact in life science.


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