KILR® Cytotoxicity CD16 Effector Cells

Driving Robust and Reproducible ADCC and T-Cell Redirection

Effector cells such as peripheral blood mononuclear cells (PBMCs) have inherent donor variability, reducing consistency and reproducibility in lot release assays. Antibody-Dependent Cell Cytotoxicity (ADCC) assays that measure cell death often have low signal-to-background (S/B) ratios and suffer from this donor variability of the PBMCs.

Eurofins DiscoverX® offers KILR CD16 Effector Cells are single donor-derived human primary cells. They are ideal for measuring target cell death using ADCC or bi-specific antibody-mediated T-cell redirection (TCR) for screening, characterization, and QC lot release of immunotherapeutics (antibody drugs). These effector cells ensure assay reproducibility with higher S/B ratios, eliminating donor variability.

Product Highlights

  • Eliminate Donor Variability – Primary effector cells (human cytotoxic T lymphocytes) from a single donor engineered to stably express CD16 (V158)
  • Fit for Long-Term QC Testing – Uniformly manufactured to ensure lot-to-lot consistency and produce high S/B ratios
  • Measure Target Cell Death – High killing capacity for implementing ADCC or TCR for lot release or characterization
Consider Eurofins DiscoverX’s custom development capabilities for custom cell lines, assays, & enzyme development.
ProductConfigurationCat. No. 
KILR® CD16 Effector Cells1 Vial97-0007-01
KILR® CD16 Effector Cells5 Vials97-0007-05
Single Donor-Derived Effector Cells

Single donor-derived cytotoxic T lymphocytes (CTLs) transfected with CD16 (FcγRIIIa-V158). KILR CD16 Effector Cells are engineered to express CD16 receptor to form an immunological synapse between the target antibody and antigen that ultimately triggers the release of perforins, granzymes, and cytokines that promote cell death when delivered to target cell cytosol. A. KILR CD16 Effector Cells showing a polyclonal population of predominantly (>98%) CD8+ cells and (B.) positive for CD3 with robust and stable expression of CD16. C. KILR CD16 Effector Cells have functional CD3 as demonstrated by the ability of cells to stimulate T-cell redirection in the presence of blinatumomab (see the section on T-cell redirection).

Evaluation of the intra- and inter-lot performance of KILR CD16 Effector Cells in a Rituximab ADCC model.

Evaluation of the intra- and inter-lot performance of KILR CD16 Effector Cells in a Rituximab ADCC model. High lot-to-lot reproducibility ensures long-term support for QC testing.

KILR ADCC Assay Workflow using KILR CD16 Effector Cells

The KILR ADCC assay workflow

The KILR ADCC assay workflow. KILR target cells of interest are engineered to stably express a housekeeping protein that is tagged with a reporter fragment (KILR reporter protein). These cells are plated, followed by the addition of the test antibody to the target cells. Following opsonization, effector cells (e.g. KILR CD16 Effector Cells) are added to the wells containing the target cells. Effector-mediated killing releases the KILR reporter protein into the media that is detected by the addition of detection reagents and results in a chemiluminescent signal that can be read on any standard benchtop luminometer.

The KILR cytotoxicity platform is based on the industry-validated Enzyme-Fragment Complementation (EFC) technology. EFC is based on two recombinant β-galactosidase (β-gal) enzyme fragments that act as an enzyme acceptor (EA) and an enzyme donor (ED). Separately, the fragments are inactive, but when combined, they form an active β-gal enzyme that hydrolyzes its substrate to produce a chemiluminescence signal.

KILR ADCC Assay Principle using KILR CD16 Effector Cells


KILR ADCC Assay Principle using KILR CD16 Effector Cells

The KILR ADCC assay principle. Target cells expressing the relevant antigen are engineered to stably express a housekeeping protein tagged with a reporter β-gal ED fragment called enhanced ProLabel® (ePL). The β-gal enzyme is inactive when the reporter fragment (also called the KILR reporter protein) is not paired with its larger β-gal EA fragment. On incubation of KILR target cells with a test antibody and appropriate effector cells (e.g. KILR CD16 Effector Cells), effector cell-mediated killing releases the tagged protein into the media. The presence of both EA and ED fragments leads to the formation of the active β-gal enzyme that hydrolyzes the substrate to give a chemiluminescent output detected on any benchtop luminometer. Overall, the single donor-derived KILR CD 16 Effector Cells are introduced to evaluate ADCC, and these effector cells are universal and can be used in any ADCC assay or T-cell redirection applications.

Obtain Robust ADCC Assay Results with Single-Donor Derived KILR CD16 Effector Cells

Improved ADCC assay performance with KILR CD16 Effector Cells. Single-donor derived KILR CD16 Effector Cells are engineered cytotoxic T lymphocytes (CTLs) derived from a single donor that are transfected with CD16 (FcγRIIIa- V158). Robust ADCC was observed with KILR CD16 effector cells than with primary cells. In KILR Raji cells treated with Rituximab, KILR CD16 Effector Cells mediate a 2.5-fold higher assay window and EMax than primary natural killer (NK) cells when used at the same effector-to-target (E:T) ratio (E:T = 10:1). The difference in assay window and EMax is even more pronounced when comparing primary PBMCs (used at an E:T = 25:1).

Achieve Significantly Larger Assay Windows to Better Analyze Antibody Activity

Large assay windows with KILR CD16 Effector Cells compared to PMBCs

Large assay windows with KILR CD16 Effector Cells compared to PMBCs. A. KILR cytotoxicity model ARH77 (CD20+) was opsonized with rituximab and then incubated with either KILR CD16 Effector Cells (E:T = 10:1) or PBMCs (E:T = 25:1). B. KILR cytotoxicity model SKOV-3 (HER2+) was opsonized with trastuzumab and then incubated with KILR CD16 Effector Cells (E:T = 12.5:1) or PBMCs (E:T = 25:1). After addition of KILR detection reagents in both assays, a 4- or 16-fold larger assay window was observed in the ARH77 or SKOV3 models, respectively, with KILR CD16 Effector Cells relative to PBMCs.

Detect Differences in Glycan Profiles for ADCC Assays

Infliximab comparability studies showing KILR CD16 Effector Cells are able to detect glycan profiles in an ADCC assay. A. Glycosylation profile of the innovator and biosimilar infliximab. The glycan profile of both molecules are consistent with a monoclonal antibody that has been expressed in mammalian cell lines. Most peaks in the biosimilar chromatogram are also observed in the innovator chromatograms although there are some variations. There are higher levels of some sialic acid species and an increase in galactosylation of G1F and G2F, and there is decreased afucosylation in the biosimilar profile that can have an impact on ADCC activity. B. ADCC activity of infliximab was subsequently assessed using purified NK cells from V/V and F/F donors, and compared with KILR CD16 Effector Cells. The following potencies were returned for the biosimilar molecule during the assay: V/V donor – 29.7%, F/F donor – 38.7%, KILR CD16 cells – 41.0%. This data is part of a comparability study conducted by BioOutsource Sartorius Stedim.

T-Cell Redirection Application using Bi-specific T-cell Engagers (BiTEs)

The KILR T-cell redirection assay application. A. Graphic illustration of theKILR t-cell redirection MOA. BiTEs are chimeric antibodies with dual specificities that target tumor cells by harnessing the power of the immune effector cells. The BiTE molecule binds to a tumor-associated antigen such as CD19. The KILR CD16 Effector Cells, which express CD3 endogenously, rapidly kills target cells via t-cell redirection. B. KILR CD16 Effector Cells were incubated with KILR Raji cells and blinatumomab (an FDA approved BiTE molecule) for 4, 5, or 6 hours at 37°C. Optimal assay window (max killing of >60%) was observed at 6 hour incubation with KILR CD16 effectors with an EC50 of 15.5 pg/mL.

Application Notes Implementing MOA-Reflective ADCC Assays using Ready-to-Use KILR Target and Effector Cells from Screening to Lot Release

The development of antibody-based therapeutics to target the killing of tumor cells has revolutionized the space of immuno-oncology and the…

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Presentations [CASSS 2017] Robust and Reproducible ADCC and T Cell Redirection with CD16-Engineered Effector Cells

DiscoverX CASSS 2017 Presentation Presented by Dr. Jane Lamerdin, Director R&D, DiscoverX May 9, 2017

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Scientific Posters Driving Robust and Reproducible ADCC and T Cell Redirection with Single Donor KILR® CD16 Effector Cells

Success of ADCC assays is highly dependent on the quality of effector cells used. However, human primary cells (such as…

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Scientific Posters A novel dye-free cytotoxicity assay to selectively measure target cell death in ADCC, CDC CAR-T or T-cell activation assays

Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism by which many Class I therapeutic antibodies, including rituximab and trastuzumab, achieve clinical…

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Scientific Posters Single Donor KILR® CD16 Effector Cells to Drive Robust and Reproducible ADCC and T Cell Redirection

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The clinical success of an ever-increasing array of biologics has led to the development of a wide spectrum of immunomodulatory…

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Webinars Driving Robust ADCC and T Cell Redirection with KILR CD16 Effector Cells

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Blogs & Articles Employing KILR Cytotoxicity Ready-to-Use Bioassays from Screening to Lot-Release Applications

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Blogs & Articles Eliminating Donor Variability in ADCC Assays – Implementation in QC Lot Release

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Blogs & Articles Developing A Simple Antibody Dependent Cell-Mediated Cytotoxicity Assay to Measure Target Cell Death

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KILR® Cytotoxicity Product Solutions

Simple, non-radioactive, & dye-free cytotoxicity assays to specifically measure target cell death

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KILR® Cytotoxicity Bioassays

Ready-to-use, cell-based bioassay kits to measure direct cell death for screening, characterization, & potency testing

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Custom Capabilities

Custom cell lines, kits, assays, & protein development capabilities optimized to fit your requirements

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