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KILR® Cytotoxicity Assay

Specifically Measure Target Cell Death in Co-Cultures

Cancer immunotherapy drugs are designed to treat cancers by boosting the body’s immune system to fight and kill the cancer cells. Assays for these drugs need to detect target cancer cell death when co-cultured with immune effector cells. Radioactive or leaky dyes make it difficult to detect target cell death in a co-culture with immune effector cells, and general cell death assays lack specificity to differentiate between cancer cell and effector cell death.  Effector cells such as peripheral blood mononuclear cells (PBMCs) used in these assays have inherent donor variability, reducing their consistency and reproducibility in lot release assays.

The KILR cytotoxicity assay eliminates these issues with a simple, non-radioactive and dye-free method to specifically measure target cell death in a co-culture with effector cells. This easy-to-use assay has broad applications for immuno-oncology (iOnc) drug development, from screening to QC lot release assays. The assay can detect cancer cell death through various mechanisms of action such as antibody dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibody dependent cell phagocytosis (ADCP), cytotoxic t-cell lymphocyte mediated death (CTL), bi-specific antibody mediated T-cell redirection, chimeric antigen receptor t-cell (CAR-T) and adoptive T-cell therapies.

Now you can rapidly develop cytotoxicity assays with the available KILR cell lines or use KILR Retroparticles to develop custom target cell lines with tremendous flexibility. KILR CD16 Effector Cells are single donor-derived human primary effector cells, which are engineered for stable CD16 expression. When used in any ADCC assay, these effector cells produce very low background, resulting in robust assay windows, making these cells well-suited for discovery, characterization, and lot release assays.
 

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.

Use KILR Effector Cells in any ADCC assay to measure cell death. Use available KILR cell lines or make your own stable KILR cell lines with retroviral particles that can transduce almost any cell line.

KILR CD16 Effector Cells

Product Configuration Cat. No.
KILR® CD16 Effector Cells, 1 Vial Primary Cells 97-0007-01
KILR® CD16 Effector Cells, 5 Vials Primary Cells 97-0007-05

KILR Retroparticles

Product Configuration Cat. No.
KILR® Retroparticles for Suspension Cells (G418) 4 Vials x 1.0 mL 97-0002
KILR® Retroparticles for Adherent Cells (G418) 4 Vials x 0.5 mL 97-0003
KILR® Retroparticles for Adherent & Suspension Cells (G418) 2 Vials x 0.5 mL (adherent cells), 2 Vials x 0.5 mL (suspension cells) 97-0004
KILR® Retroparticles for Adherent Cells (Hygromycin B) 4 Vials x 0.5 mL 97-0005
KILR® Retroparticles for Suspension Cells (Hygromycin B) 4 Vials x 1.0 mL 97-0006
KILR® Retroparticles for Suspension & Adherent Cells (Hygromycin B) 2 Vials x 0.5 mL (adherent cells), 2 Vials x 0.5 mL (suspension cells) 97-0008

KILR Target Cell Lines

Product Configuration Cat. No.
KILR® ARH-77 Cell Line Stable Cell Lines 97-1001C017
KILR® SKBR3 Cell Pool Cell Pool 97-1002P018
KILR® H322 Cell Pool Cell Pool 97-1003P020
KILR® NCI-N87 Cell Pool Cell Pool 97-1004P021
KILR® A549 Cell Pool Cell Pool 97-1005P015
KILR® Ramos Cell Pool Cell Pool 97-1006P022
KILR® SKOV3 Cell Pool Cell Pool 97-1007P023
KILR® NCI-H292 Cell Pool Cell Pool 97-1008P024
KILR® Daudi Cell Pool Cell Pool 97-1009P025
KILR® U2OS-EGFR Cell Line Stable Cell Lines 97-1010C003
KILR® THP-1 Cell Pool Cell Pool 97-1011P014
KILR® Raji Cell Pool Cell Pool 97-1012P026
KILR® WIL2-S Cell Pool Cell Pool 97-1013P027
KILR® MCF7 Cell Pool Cell Pool 97-1014P028
KILR® MOLT-4 Cell Pool Cell Pool 97-1015P029
KILR® SK-MEL-28 Cell Pool Cell Pool 97-1017P031
KILR® U118-MG Cell Pool Cell Pool 97-1018P032
KILR® EL4 Cell Pool Cell Pool 97-1019P033
KILR® Jurkat Cell Pool Cell Pool 97-1020P019
KILR® A498 Cell Pool Cell Pool 97-1021P034
KILR® MDA-MB-231 Cell Pool Cell Pool 97-1023P036
KILR® Hut78 Cell Pool Cell Pool 97-1024P037
KILR® T2 Cell Pool Cell Pool 97-1025P038
KILR® RPMI 8226 Cell Pool Cell Pool 97-1026P039
KILR® CCRF-CEM Cell Pool Cell Pool 97-1027P040
KILR® 4T1 Cell Pool Cell Pool 97-1028P041
KILR® U2OS-PD-L1 Cell Line Stable Cell Lines 97-1029C003
KILR® K562 Cell Pool Cell Pool 97-1030P042
KILR® HT-1080 Cell Pool Cell Pool 97-1031P043
KILR® HepG2 Cell Pool Cell Pool 97-1032P044
KILR® SK-MEL-5 Cell Pool Cell Pool 97-1033P030

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
 
  1. Measurement of a biologically relevant ADCC endpoint which is target cell death
  2. Get a specific measurement of target cell death with a sensitive and robust signal
  3. 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
 
  1. Sensitive measurement of target cell death
  2. 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.
 
  1. Industry’s first plate-based high-throughput ADCP assay
  2. Measure biologically relevant ADCP endpoint, which is target death
  3. Specific measurement of target cell death
  4. 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