Advantages Of The KILR Assay
- Unparalleled Specificity – Signal only from dead target cells
- Easy-to-Use – Simple add and read protocol with chemiluminescent readout
- Exquisite Sensitivity – Detect as few as 75 dead cells with high reproducibility
- Ultimate Flexibility – Ability to run cytotoxicity assay from 30 minutes to 72 hours
- Eliminate Donor Variability – Primary effector cells from a single donor
Highly Specific – KILR detection protein only present in target cells
Target cells expressing the receptor antigen of choice can be engineered to stably express a protein tagged with enhanced ProLabel (ePL), a β-gal reporter fragment, using the KILR Retroparticles. When the stable target cell line is used in a cytotoxicity assay, and its membrane is compromised due to cell death, it will release the tagged protein into the media. We can detect this KILR reporter protein in the media by the addition of detection reagents containing the enzyme acceptor (EA) fragment of the β-gal reporter. This leads to the formation of the active β-gal enzyme which hydrolyzes the substrate to give a chemiluminescent output, detected on any bench top luminometer.
Easy to use – Simple, one-step add and read protocol
A simple non-radioactive, add and read protocol with a chemiluminescent output that can be read on any benchtop luminometer. Using the KILR assays, you can eliminate the need to load target cells prior to every experiment, the use of radioactivity, and reduce the number of steps, increasing the efficiency of the lab.
Highly Sensitive – Robust detection of molecules with low-level toxicity
KILR H322 cells were plated at various densities with primary human PBMCs and Cetuximab, an anti-EGFR drug for colorectal cancers, to measure ADCC response. ADCC is a known mechanism of action for Cetuximab, where the antibody activates immune cells to kill target cancer cells. Target cell death is reported as % Lysis, a ratio of experimental signal to total signal generated when the cells are lysed with detergent. The data on the left shows we can robustly detect 3% Lysis in the KILR H322 cells when a total of 2500 KILR cells are plated in the well, indicating that we have detected the death of 75 cells inside the well. This exquisite sensitivity of the assay demonstrates its value in applications such as screening and lead optimization, where sensitivity is critical to identify and optimize lead drug candidates.
A. The KILR CD16 Effector Cells are added to the plated target cells expessing the receptor antigen, which have been engineered to stably express a housekeeping protein that is tagged with enhanced ProLabel® (ePL), a β-gal enzyme fragment using the KILR Retroparticles. When the stable target cell line is used in a cytotoxicity assay, and its membrane is compromised due to cell death, it will release the tagged reporter protein into the media. We can detect this reporter protein by the addition of detection reagents containing the enzyme acceptor (EA) fragment of the β-gal enzyme. This leads to the formation of the active β-gal enzyme, which hydrolyzes the substrate to give a chemiluminescent output, detected on any bench top luminescence reader.
B. In the figure the well on the left contains healthy, intact target cells that are alive in the presence of immune effector cells. When detection reagents are added to the well, we cannot detect chemiluminescence as the reporter protein does not leak out through an intact cell membrane into the media. Alternatively, in the well on the right, the target cancer cells are killed by the KILR CD16 Effector Cells, releasing the reporter protein into the media. Addition of the detection reagents leads to the recognition of the reporter protein and generation of the chemiluminescent signal that is proportional to the number of dead cells. Death of any other cell type, including the KILR CD16 Effector Cells present within the co-culture will not affect the assay output, giving the KILR Cytotoxicity assay an unparalleled specificity to detect target cell death within a co-culture system.
Example of an ADCC Assay with KILR Cell Lines and KILR CD16 Effector Cells
Two KILR cytotoxicity models, A. ARH77 (CD20+) and B. SKOV-3 (HER2+) were opsonized with the appropriate antibody, then incubated with primary PBMCs (E:T = 10:1 or 12.5:1, respectively) for 3 hours, follwed by the addition of KILR Detection Reagent. A 4-fold larger assay window was observed in the CD20 model with KILR CD16 Effector Cells relative to PBMCs, while an even larger (16-fold) improvement in assay window was observed with the more difficult to kill SKOV-3 cell model.
The KILR assays have broad applications, some of which have been outlined below.
- Antibody Dependent Cell-mediated Cytotoxicity (ADCC)
- Complement Dependent Cytotoxicity (CDC)
- Antibody Dependent Cellular Phagocytosis (ADCP)
- Bi-specific antibody mediated T-cell redirection
- Chimeric Antigen Receptor T-Cell (CAR-T)
- Antibody Drug Conjugate (ADC)
Antibody Dependent Cell-Mediated Cytotoxicity (ADCC)
Antibody Dependent Cell-mediated Cytotoxicity (ADCC) is a mechanism of cell-mediated immune defense. The signaling events involve the Fab portion of the antibody binding to a specific antigen on a target cell and the FcgRIII (or CD16) on the effector cells binds to the exposed Fc portion of the antibody, which activates the cell, leading to release of various granzymes and cytokines and causing cell death. With the KILR ADCC assay, antibody-mediated cell death is monitored through the release of cellular protein from dying or lysed cells and measured using a simple add & read assay. On the left we have data demonstrating Cetuximab (Erbitux®)-mediated cytotoxicity in U-2 OS cells expressing EGFR. The use of the KILR assay offers several advantages compared to existing methods
- Measurement of a biologically relevant ADCC endpoint which is target cell death
- Get a specific measurement of target cell death with a sensitive and robust signal
- Easy-to-use protocol eliminates complicated dye-loading steps
Complement Dependent Cytotoxicity (CDC)
Complement dependent cytotoxicity (CDC) is an immune mediated defense function. The antibodies bind to the membrane antigens on the cell surface of the target cancer cells. Normal human complement binds to and is activated by the Fc region of the antibody causing a complement cascade and the induction of a membrane attack complex. This causes target cell death through lysis. On the left, we present data demonstrating Rituximab-mediated CDC using rabbit complement with ARH-77 cells. The use of the KILR assay offers several advantages compared to existing methods
- Sensitive measurement of target cell death
- Easy to use protocol
Antibody Dependent Cellular Phagocytosis (ADCP)
ADCP is a mechanism of immune defense where macrophages engulf and destroy cells that have been coated with certain types of antibodies. The Fab portion of the antibody drug binds to a cell-surface antigen on the target cell and the FcγRIIa on the macrophages bind to the exposed Fc portion of the antibody. This activates the macrophage to engulf, kill and destroy the target cancer cells which contain the KILR ePL construct. The KILR ADCP assay is the first plate-based ADCP assay that truly measures end-point ADCP by measuring the drop in total chemiluminescence when compared to a no antibody or isotype control, as the destruction of the target KILR cells leads to a destruction of the ePL protein tag. The only signal measured in this assay comes from the KILR target cells, making this a highly specific readout. Example data on the left demonstrate the robust performance of this highly-specific, simple, plate-based ADCP assay using primary macrophages.
- Industry’s first plate-based high-throughput ADCP assay
- Measure biologically relevant ADCP endpoint, which is target death
- Specific measurement of target cell death
- Easy-to-use protocol eliminates complicated and expensive FACS assays
Bi-Specific Antibody Mediated T Cell Redirection (TCR)
Therapeutic bi-specific antibody engages and activates cytotoxic T lymphocytes and can redirect T cell cytotoxicity towards tumor cells (or other target cells of interest) by cross-linking the T cell receptor complex on the surface of T cells with tumor-specific antigens or other target antigens of interest.
Example data on the left demontrate a larger assay window and rapid killing kinetics of the KILR CD16 Effector Cells.
Measure biologically-relevant redirected T cell cytotoxicity
Get results fast with potent effector cells
Eliminate donor variability with single donor-derived effector cells