APPLICATION NOTE

Quantify Secreted T Cell
Activation Markers in90 Minuteswith Simple PlexTM Automated ELISAs

Introduction
Although Chimeric Antigen Receptor (CAR) T cells havegreat therapeutic utility, the manufacturing workflow iscomplex and stringent regulatory requirements must bemet. CAR T cells must be thoroughly characterized in vitro
before infusion into the patient. Bio-Techne is pleased tooffer Simple Plex T Cell activation marker assays (TABLE 1)
to support cell therapy development and manufacturing
scientists. Simple Plex assays on the Ella platform offera rapid and precise assessment of secreted T cell
activation markers to boost cell therapy development andmanufacturing efficiency.

T CELL ACTIVATION MARKERS
Granzyme B

IFN-γ

IL-2

TNF-α

Perforin
TABLE 1. Simple Plex T Cell Activation Marker Assays

In this application note, we use TcBusterTM, Bio-Techne’snovel non-viral gene delivery technology, to generate CD19
CAR T cells and validate the use of Ella as a T Cell activation
marker assay platform. Read on to learn more.

CD19 CAR T Cells Lyse CD19+ Cancer Cells
Only
The first experiment was designed to test CD19 CAR T celllysing efficacy. T cells were engineered to express anti-CD19
CAR using TcBusterTM, a next-generation non-viral genedelivery system. CD19 CAR T cells were co-cultured withluciferase-expressing cancer cells that co-express CD19(Nalm-6) or do not (K562). After 24 hours of co-culture, cellsupernatants were combined with luciferin and assayed
for luciferase expression. Cellular lysis was enhanced in theNalm-6 co-culture but not the K562 co-culture. Lysis wasequivalent to non-CD 19 transposed control T cells in theK562 culture. This data indicates that the anti-CD19 CARexpressing T cells lysed the CD19+Nalm-6 cells but not theCD19– K562 cells (FIGURE 1).

A.

B.

FIGURE 1. Cell Lysis was observed in NALM-6 co-cultures but not K562 co-cultures. CD19 T cells and Nalm-6 (A) or CD19 T cells and K562(B) cancer cells wereco-cultured at varying ratios (10:1, 5:1, 2.5:1, 1.25:1, 0.63:1, or 0.31:1) for 24 hours. Data were normalized to relative light units (RLUs) in the cancer cell-only wells.
Bar graphs = mean ± SD (n=3), ****P<0.0001, **P< 0.01.

Simple Plex Assays Rapidly Quantify T Cell Activation Marker Secretion
Next, we wanted to assess T cell activation marker secretion. Anti-CD19 CAR expressing T cells were co-cultured with Nalm-6
or K562 cancer cells at a ratio of 5:1 for 24 hours. After co-culturing, supernatants were collected and centrifuged at 500 x gfor 5 min to remove cell debris. Supernatants were assayed in 90 minutes using the following Simple Plex T Cell Marker assays:Granzyme B, TNF-α, IFN-γ, and IL-2. FIGURE 2 indicates that T cell marker secretion was robustly elevated relative to controls in
the CD19 CAR T/Nalm-6 co-cultures only.

FIGURE 2. T cell activation markers are robustly elevated when CD19 CAR T cells are co-cultured with CD19+ Nalm-6 cancer cells. T cell activation markers
Granzyme B, TNF-α, IFN-γ, and IL-2 were assayed in parallel using a 32x4 Multianalyte cartridge on the Ella platform. Bar graphs = mean ± SD (n=3).

2Simple Plex Immunoassays Quantify Perforin Release in 90 Minutes
To evaluate Perforin secretion, supernatants from various ratios of Nalm-6 and K562 co-cultures were collected and analyzed
using the Simple Plex Perforin assay. As seen in FIGURE 3, all CD19+/Nalm-6 co-culture supernates secreted more Perforin thancontrol co-cultures. Only the highest (10:1) ratio CD19 CAR T cell/K562 secreted more Perforin relative to control co-cultures.

A.

B.

FIGURE 3. All Nalm-6 cocultures exhibited statistically significant increase in Perforin secretion. Bar graphs = mean ± SD (n=3), ****P<0.0001, **P< 0.01.

Conclusion
The rationale for this series of experiments was to demonstrate the value of Simple Plex immunoassays in celltherapy development and manufacturing. The findings of this study are visualized in FIGURE 4. CD19+Nalm-6
cells are susceptible to CD19 CAR T cell-mediated lysis. The secretion of Perforin and T cell activation markers aredramatically enhanced when these cancer cells are co-cultured with CD19 CAR T cells. Conversely, CD19- K562
cells are not susceptible to CD19 CAR T lysis. T Cell activation markers and Perforin were not robustly secreted after
co-culture with CD19 CAR T cells.

FIGURE 4. Cytokine and Perforin secretion, as well as cellular lysis, is observed in CD19+ Nalm-6/ CAR T co-cultures (A) but not in CD19- K562/ CAR T co-cultures (B).

3

Easy To Use Simple Plex Assays Get You
Accurate Data in 90 Minutes.

Quantikine™ T Cell Activation ELISA Kits
These ready-to-use ELISA Kits are the most cited in themarket.

We have demonstrated that Simple Plex multianalyte assays
provide high-quality, quantitative data in just 90 minutes with
unparalleled flexibility and ease of use. Simply load yoursamples and press start; no additional reagents are needed.
Simple Plex immunoassay benefits include a built-in factory
standard curve, up to 4 logs of dynamic range, and 21 CFRPart 11 compliance for cell therapy manufacturing processes.
Simple Plex assays are vertically integrated with R&D Systems
antibodies to deliver the reproducible data you can trust.

Granzyme B
IFN-γ
IL-2
IL-8/CXCL8
TNF-αCell and Gene Therapy Development andManufacturing Resources

Simple Plex T Cell Activation Marker Assays

TcBusterTM Non-Viral Gene Engineering Services
CloudzTM Cell Activation Kits
GMP Proteins for Therapeutic Manufacturing
Learn About Bio-Techne’s GMP Capabilities

Get accurate and reproducible data in 90 minutes.
Meet Ella and Request Pricing
Granzyme B
IFN-γ
IL-2
IL-8/CXCL8
Perforin
TNF-α

R&D Systems™ Novus Biologicals™

Tocris Bioscience™

ProteinSimple™

ACD™

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