Applications & Products / KILR Cytotoxicity Assays
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Applications & Products

KILR® Cytotoxicity Product Solutions

Specifically Measure Direct Target Cell Death

Cytotoxicity assays are required to measure target cell death via several mechanisms of actions (MOAs). These MOAs include 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. Such assays are designed to capture a therapeutics’ ability to cause targeted cell death via one or more primary MOAs, and to predict or mimic a physiological response. Cancer immunotherapy (immune-oncology) drug therapeutics 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 or in the presence of the complement system.

 

Commonly used assays for measuring cytotoxicity include reporter gene and fluorescent dye-based assays. However, these assays suffer from disadvantages, including variability induced by effector cells. Reporter gene assays only predict the MOA potential and typically require a bridging assay (as needed for ADCC) to quantify target cell death. Fluorescent and radioactive dye-based assays pose safety challenges and run the risk of the spontaneous release of dyes from the cells, causing leaks, thus impacting the accuracy and reproducibility of the data. Effector cells such as peripheral blood mononuclear cells (PBMCs) used in these assays tend to have inherent donor variability, reducing their consistency and reproducibility in lot release assays.

 

Eurofins DiscoverX® KILR (Killing Immune-Lysis Reaction) cytotoxicity assay platform provides a simple, non-radioactive, and dye-free method to specifically measure target cell death that avoids the drawbacks of the other commonly used cytotoxicity assays. This homogeneous, plate-based assay platform has broad applications from screening to QC lot-release testing, particularly for immuno-oncology drug development applications such as ADCC, ADCP, CDC, CAR-T, and T-cell redirection. The flexibility of the KILR platform allows you to utilize stable cell lines or cell pools in relevant tumor models. To provide additional flexibility and ease-of-use, accelerate your drug development programs by several months with ready-to-use bioassay format, or eliminate donor-to-donor variability observed with PBMCs with the KILR CD16 Effector Cells.

 

Cytotoxicity BioassaysCD16 Effector Cells

 

 

Highlights of KILR Cytotoxicity Assays

  • Ultra-Specific & Sensitive – Detect cytotoxicity only from lysed target cells and as few as 75 dead cells with high reproducibility
  • Ultimate Flexibility – Ability to use with different effector cell types and run cytotoxicity assays from 30 minutes to 72 hours
  • Biologically Relevant – Reflective of the true MOA of the therapeutic or ligand
  • Multiple Applications – Analyze ADCC, ADCP, CDC, CAR-T, ADC, CTL mediated death, bi-specific antibody-mediated T-cell redirection, and adoptive T-cell therapies

 

High Assay Specificity

High Assay Specificity

 

The KILR ADCC assay response is highly specific. A. Cetuximab mediates an ADCC response in U2OS cells over-expressing EGFR, but not in native U2OS cells that have low levels of endogenous EGFR. B. A highly specific ADCC response is observed with Cetuximab targeting U2OS cells expressing EGFR as opposed to that of Rituximab that targets CD20 that results in no ADCC. NK = Natural Killer. E:T = Effector Cells:Target Cells. S/B = signal-to-background.

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Compatible with Multiple Effector Cell Types

Compatible with Multiple Effector Cell Types

 

KILR ADCC Assays are compatible with multiple effector types. A. ADCC using primary PBMCs from 3 different donors in the KILR SKBR3 model B. ADCC with two HER2-targeting antibodies using primary NK cells in the KILR SKBR3 model. C. ADCC with Trastuzumab using and engineered NK-92 cell line as effector cells, with the NK-92-resistant SKOV3 cell pool. NK = Natural Killer. E:T = Effector Cells:Target Cells. S/B = signal-to-background.

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Create Your Own Cell-Based Cytotoxicity Assays in Any Dividing Cell Type

Create Your Own Cell-Based Cytotoxicity Assays in Any Dividing Cell Type

 

Generate cell-based cytotoxicity assays in any dividing cell type. Using Enzyme Fragment Complementation (EFC), which is based on a split β-galactosidase (β-gal) enzyme you can create your own cytotoxicity assays. Target cells expressing the receptor antigen of choice can be engineered to stably express a protein (housekeeping protein) tagged with the small β-gal enzyme donor (called the enhanced ProLabel (ePL) or β-gal reporter fragment) using KILR Retroparticles. When the stable target cell line is used in an EFC-based cytotoxicity assay (e.g., ADCP, ADCC, CDC), the EFC reaction can be measured with the addition of the large β-gal enzyme acceptor (EA) fragment that creates the active β-gal enzyme. With the addition of hydrolyzing substrate, a chemiluminescent output can be detected on any bench top luminometer.

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Key Resources

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Choose from a list of different products offered within the KILR platform to support your immuno-oncology drug discovery and development journey. These products include bioassay kits, effector cells, stable cell lines, cell pools, and retroparticles for use in screening applications and relative potency testing in lot-release programs. Consider ready-to-use bioassays with different immune effectors cells like PBMCs, NK cells, or KILR Effector Cells for potency or lot-release testing. Use KILR Effector Cells in any ADCC assay to measure target cell death. With stable cell lines and cell pools, select the relevant tumor model and study the Fc-mediated effector functions of your therapeutic antibody. Or, generate your own stable KILR cell pools/lines with retroviral particles that can transduce almost any cell line.

 

To obtain custom KILR cell lines, cell pools, or assays, contact custom development capabilities.

Product List

 

KILR Bioassay Kits

Product Configuration Cat. No.
KILR® Raji ADCC Bioassay Kit 2-Plate 97-1012Y026-00169
KILR® Raji ADCC Bioassay Kit 10-Plate 97-1012Y026-00170
KILR® Raji ADCP Bioassay Kit 2-Plate 97-1012Y026-00179
KILR® Raji ADCP Bioassay Kit 10-Plate 97-1012Y026-00180
KILR® Daudi ADCC Bioassay Kit 2-Plate 97-1009Y025-00171
KILR® Daudi ADCC Bioassay Kit 10-Plate 97-1009Y025-00172
KILR® Daudi ADCP Bioassay Kit 2-Plate 97-1009Y025-00177
KILR® Daudi ADCP Bioassay Kit 10-Plate 97-1009Y025-00178

 

Request qualification data set for KILR® Raji ADCC Bioassay Kit using rituximab.

 

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KILR Effector Cells

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

 

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KILR Cell Lines and Cell Pools

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
KILR® COLO-205 Cell Pool Cell Pool 97-1034P045
KILR® U2OS PD-L2 Cell Line Stable Cell Lines 97-1036C003
KILR® Jurkat PD-1 Cell Line Stable Cell Lines 97-1037C019
KILR® Jurkat LAG3 Cell Line Stable Cell Lines 97-1038C019
KILR® HL-60 Cell Pool Cell Pool 97-1039P046
KILR® HCT-116 Cell Pool Cell Pool 97-1040P047
KILR® BT-474 Cell Pool Cell Pool 97-1042P049
KILR® DU-145 Cell Pool Cell Pool 97-1043P050
KILR® PANC-1 Cell Pool Cell Pool 97-1044P051
KILR® MM-1R Cell Pool Cell Pool 97-1045P052
KILR® Jurkat TIM3 Cell Line Stable Cell Lines 97-1046C019
KILR® SR Cell Pool Cell Pool 97-1047P053

 

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KILR Retroparticles

Product Configuration Cat. No.
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® Retroparticles for Suspension Cells (G418) 4 Vials x 1.0 mL 97-0002

 

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Custom and Toolbox Products

  • Custom Capabilities – Custom cell lines, cell pools, and assays capabilities optimized to fit your requirements
  • Toolbox Products – Complete set of parental cell lines, expression and cloning vectors, kits, and retroparticles to build your own stable cell lines and cell-based assays

 

The KILR 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

KILR ADCC Assay Principle

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, 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.

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KILR ADCP Assay Principle

KILR ADCP Assay Principle

The KILR ADCP assay principle. ADCP measures antibody-dependent phagocytosis of target cells, typically mediated by primary human macrophages. KILR target cells, containing the KILR reporter protein (including the β-gal ED fragment also called ePL), undergo phagocytosis by effector cells (e.g. macrophages differentiated from monocytes) leading to lysosomal degradation of the KILR reporter protein. In this assay, the KILR target cells are opsonized with the antibody of interest, then co-cultured with the effector cells. Macrophages perform phagocytosis of the target cells. All non-phagocytosed cells are lysed and the detection reagent containing the complementing β-gal EA fragment is added to the lysed cells. Complementation of ED and EA with the addition of substrate results in the detection of the non-phagocytosed KILR protein present. A high chemiluminescent signal indicates minimum killing, while a low signal correlates to higher phagocytosis or increased ADCP activity. Note: other immune effector cells can also be used for the ADCP assay.

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KILR CDC Assay Principle

KILR CDC Assay Principle

The KILR CDC assay principle. CDC is mediated by the C1 complex that initiates deposition of C3b fragments on target cells. The C3b in conjunction with C4b and C2a forms the C5 convertase enzyme that cleaves C5 into C5a and C5b. C5b then participates in the formation of the membrane attack complex or MAC (C5b-C6-C7-C8-C9). MAC is responsible for the cytolytic function of the complement system resulting in subsequent target cell lysis. The KILR reporter protein (including the β-gal ED fragment also called ePL) is stably expressed in the target cells and released into the media when these target cells undergo lysis as a result of MAC formation. The amount of total KILR reporter protein is detected by the addition of detection reagents containing the β-gal EA fragment. Complementation of ED and EA facilitates detection of the remaining amount of KILR protein present, and with the addition of subtrate, hydrolysis occurs and a chemiluminescent signal can be detected on any bench top luminometer.

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KILR ADCC Application

KILR ADCC Application

The KILR ADCC assay application. A. Rituximab-mediated ADCC in four different CD20+ B-lymphoblast KILR models (ARH-77, Daudi, Ramos, and WIL2-S) using primary PBMCs. B. ADCC mediated by the anti-CD38 therapeutic antibody, Daratumumab, in the KILR Raji cell model using engineered effector cells (KILR CD16 effector cells). C. ADCC mediated by the anti-CD33 therapeutic antibody, Gemtuzumab (approved for treatment of AML), in the KILR HL-60 cell model using primary human PBMCs. This data demonstrates that KILR ADCC assays can be used to evaluate diverse cancer models. E:T = Effector Cells:Target Cells.

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KILR ADCP Application

KILR ADCP Application

The KILR ADCP assay application. A. Robust ADCP observed with trastuzumab with KILR NCI-N87 cell pool (a HER2+ solid tumor model). B. A robust dose-dependent increase in % ADCP was observed with increasing concentrations of rituximab. An EMAX value of 83% was obtained with a low EC50 of 2.4 ng/mL. E:T = Effector Cells:Target Cells.

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KILR CDC Application

KILR CDC Application

The KILR CDC assay application. CDC activity was assessed with KILR Raji bioassay cells. This assay responds specifically to rituximab and exhibits a wide dynamic range as indicated by the % lysis of the KILR target cells.

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

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

The KILR T-cell redirection assay application. KILR CD16 Effector Cells were incubated with KILR Raji cells and blinatumomab (an FDA approved BiTE molecule) for 4 through 7 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.

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