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.

Product Highlights

  • 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

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.

ProductConfigurationCat. No.
KILR® NCI-N87 ADCC Bioassay Kit2-Plate97-1004Y021-00173
KILR® NCI-N87 ADCC Bioassay Kit10-Plate97-1004Y021-00174
KILR® Raji ADCC Bioassay Kit2-Plate97-1012Y026-00169
KILR® Raji ADCC Bioassay Kit10-Plate97-1012Y026-00170
KILR® Raji ADCP Bioassay Kit2-Plate97-1012Y026-00179
KILR® Raji ADCP Bioassay Kit10-Plate97-1012Y026-00180
KILR® Daudi ADCC Bioassay Kit2-Plate97-1009Y025-00171
KILR® Daudi ADCC Bioassay Kit10-Plate97-1009Y025-00172
KILR® Daudi ADCP Bioassay Kit2-Plate97-1009Y025-00177
KILR® Daudi ADCP Bioassay Kit10-Plate97-1009Y025-00178

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

ProductConfigurationCat. No. 
KILR® CD16 Effector Cells1 Vial97-0007-01
KILR® CD16 Effector Cells5 Vials97-0007-05
ProductConfigurationCat. No. 
KILR® ARH-77 Cell LineStable Cell Lines97-1001C017
KILR® SKBR3 Cell PoolCell Pool97-1002P018
KILR® H322 Cell PoolCell Pool97-1003P020
KILR® NCI-N87 Cell PoolCell Pool97-1004P021
KILR® A549 Cell PoolCell Pool97-1005P015
KILR® Ramos Cell PoolCell Pool97-1006P022
KILR® SKOV3 Cell PoolCell Pool97-1007P023
KILR® NCI-H292 Cell PoolCell Pool97-1008P024
KILR® Daudi Cell PoolCell Pool97-1009P025
KILR® U2OS-EGFR Cell LineStable Cell Lines97-1010C003
KILR® THP-1 Cell PoolCell Pool97-1011P014
KILR® Raji Cell PoolCell Pool97-1012P026
KILR® WIL2-S Cell PoolCell Pool97-1013P027
KILR® MCF7 Cell PoolCell Pool97-1014P028
KILR® MOLT-4 Cell PoolCell Pool97-1015P029
KILR® SK-MEL-28 Cell PoolCell Pool97-1017P031
KILR® U118-MG Cell PoolCell Pool97-1018P032
KILR® EL4 Cell PoolCell Pool97-1019P033
KILR® Jurkat Cell PoolCell Pool97-1020P019
KILR® A498 Cell PoolCell Pool97-1021P034
KILR® MDA-MB-231 Cell PoolCell Pool97-1023P036
KILR® Hut78 Cell PoolCell Pool97-1024P037
KILR® T2 Cell PoolCell Pool97-1025P038
KILR® RPMI 8226 Cell PoolCell Pool97-1026P039
KILR® CCRF-CEM Cell PoolCell Pool97-1027P040
KILR® 4T1 Cell PoolCell Pool97-1028P041
KILR® U2OS-PD-L1 Cell LineStable Cell Lines97-1029C003
KILR® K562 Cell PoolCell Pool97-1030P042
KILR® HT-1080 Cell PoolCell Pool97-1031P043
KILR® HepG2 Cell PoolCell Pool97-1032P044
KILR® SK-MEL-5 Cell PoolCell Pool97-1033P030
KILR® COLO-205 Cell PoolCell Pool97-1034P045
KILR® U2OS PD-L2 Cell LineStable Cell Lines97-1036C003
KILR® Jurkat PD-1 Cell LineStable Cell Lines97-1037C019
KILR® Jurkat LAG3 Cell LineStable Cell Lines97-1038C019
KILR® HL-60 Cell PoolCell Pool97-1039P046
KILR® HCT-116 Cell PoolCell Pool97-1040P047
KILR® BT-474 Cell PoolCell Pool97-1042P049
KILR® DU-145 Cell PoolCell Pool97-1043P050
KILR® PANC-1 Cell PoolCell Pool97-1044P051
KILR® MM-1R Cell PoolCell Pool97-1045P052
KILR® Jurkat TIM3 Cell LineStable Cell Lines97-1046C019
KILR® SR Cell PoolCell Pool97-1047P053
ProductConfigurationCat. No.
KILR® Retroparticles for Adherent Cells (G418)4 Vials x 0.5 mL97-0003
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 mL97-0005
KILR® Retroparticles for Suspension Cells (Hygromycin B)4 Vials x 1.0 mL97-0006
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 mL97-0002
The KILR cytotoxicity assay platform specifically measures the killing of antigen-expressing target cells in co-culture with effector cells.

The KILR cytotoxicity assay platform specifically measures the killing of antigen-expressing target cells in co-culture with effector cells. This robust and flexible platform can be used for the quantitation of direct cell death mediated through multiple MOAs such as ADCC, ADCP, CDC, T-cell redirection, and CAR-T.

The KILR ADCC assay response is highly specific.

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.

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.

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.

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.

KILR ADCP Assay Workflow

The KILR ADCP assay workflow. CD14+ Monocytes are thawed and plated with the addition of 50 ng/mL of mCSF. Following 7 days of incubation, KILR target cells of interest, expressing the KILR reporter protein and a test antibody, are added to the plate wells containing the monocytes, now differentiated into macrophages. The macrophages phagocytose the opsonized KILR target cells leading to the degradation of the KILR reporter protein. The addition of detection reagent causes cell lysis. Complementation of two β-gal fragments with the addition of substrate results in a dose-dependent decrease in chemiluminescent signal with increasing concentrations of the test antibody. Note a shorter protocol is available and included within the ADCP application note.

KILR CDC Assay Workflow

The KILR CDC 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 addition of test antibody to target cells. Following opsonization, appropriate complement is added to the wells containing the target cells. Complement-mediated killing releases the KILR reporter protein into the media, which is detected by the addition of detection reagents and results in a chemiluminescent signal that can be read on any standard bench top luminometer.

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.

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.

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.

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.

The KILR ADCC assay application.

The KILR ADCC assay application. A. Graphic illustration of the KILR ADCC MOA. B. Rituximab-mediated ADCC in four different CD20+ B-lymphoblast KILR models (ARH-77, Daudi, Ramos, and WIL2-S) using primary PBMCs. C. ADCC mediated by the anti-CD38 therapeutic antibody, Daratumumab, in the KILR Raji cell model using engineered effector cells (KILR CD16 effector cells). D. 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.

The KILR ADCP assay application

The KILR ADCP assay application. A. Graphic illustration of the KILR ADCP MOA. B. Robust ADCP observed with trastuzumab with KILR NCI-N87 cell pool (a HER2+ solid tumor model). C. 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.

The KILR CDC assay application

The KILR CDC assay application. A. Graphic illustration of the KILR CDC MOA. B. 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.

The KILR T-cell redirection assay application

The KILR T-cell redirection assay application. A. Graphic illustration of the KILR 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

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Application Notes A Simple & Powerful Cell-Based Assay to Quantify Specific Target Cell Death by Antibody-Dependent Cellular Phagocytosis (ADCP)

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Application Notes CAR-T Cell Screening in Tumor Spheroids using Corning® Spheroid Microplates and the KILR® Cytotoxicity Assay

Audrey B. Bergeron, B.A. and Hannah J. Gitschier, M.S. Corning Incorporated, Life Sciences Kennebunk, Maine

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Scientific Posters Quantify Antibody-Dependent Cell Phagocytosis (ADCP): Application of a Robust, Non-Radioactive KILR Cytotoxicity Platform

Cell-based assays play an important role in determination of mechanism of action (MOA) for therapeutic antibodies, particularly when evaluating their…

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Scientific Posters Quantify Diverse Immune Cell-Mediated Killing Mechanisms: Applications of a Robust, Non-Radioactive KILR Cytotoxicity Platform

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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

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Scientific Posters CAR-T Cell Screening in Tumor Spheroids Using Corning Spheroid Microplates

Chimeric antigen receptor (CAR)-T cells, which are engineered to recognize target cell surface antigens expressed on tumor cells, have shown…

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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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Scientific Posters Specifically Measure Target Cell Death in a Co-Culture with Immune Cells Through a Non-Radioactive, Dye-Free Cytotoxicity Assay

Following the recent regulatory approvals of a number of checkpoint inhibitor therapeutics, development of a wide array of immunomodulatory therapeutic…

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Videos Accelerating Immuno-Oncology Drug Discovery with MOA-Reflective, Functional Cell-Based Assays

Listen to the US PEGS 2023 conference talk by Venkatesh Chari, Ph.D. on MOA-reflective, functional cell-based assays (cell lines and…

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Videos KILR Platform for Cytotoxicity Applications

Learn how KILR® cytotoxicity assays provide a simple, non-radioactive and dye free method to specifically measure target cell death in…

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Videos Single Donor-Derived KILR CD16 Effector Cells for ADCC and T Cell Redirection Applications

Learn how KILR CD16 effector cells ensure assay reproducibility with higher signal-to-background ratios, without worrying about donor variability. PBMC effector…

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Videos Custom Development Capabilities

Learn about Eurofins DiscoverX cell line engineering, assay development, and recombinant enzyme production custom capabilities. Find out more about cell…

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Target Lists Products Target List

Comprehensive Eurofins DiscoverX product list to enable your drug discovery and development programs focused on checkpoint receptors, cytokine receptors/interleukins, cytotoxicity,…

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Target Lists Cell-based Assays for Biologics Development Programs

Accelerate your biologics development from discovery to QC lot release with cell-based assays. Comprehensive list of bioassay kits, cell lines,…

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Webinars Cytotoxicity Bioassays – Measuring Antibody-Mediated Direct Cell Death

The webinar focusses on measuring antibody-mediated cell death using DiscoverX’s KILR Bioassays, and how they can be used to measure…

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

PBMC effector cells have inherent donor variability, reducing their consistency and reproducibility in lot release assays. Antibody-Dependent Cell Cytotoxicity (ADCC)…

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Webinars Implementing ADCC assays in QC: When donor variability isn’t an option

Inherent donor variability of immune effector cells presents a significant challenge for developing a robust and reproducible ADCC assays for…

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Case Studies Implementing Robust & Reproducible KILR® Ready-to-Use Bioassays from Screening to Lot Release: Applications for Measuring Direct Cytotoxic Cell Death

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

Therapeutic antibodies that can elicit different applications of cell-mediated cytotoxicity, such as Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) and many other effector…

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

Class I therapeutic antibodies achieve their clinical efficacy by binding to their target antigen and through Fragment crystallisable region (Fc)…

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Blogs & Articles Do-It-Yourself: Benefits and Challenges of Creating Your Own Cell-Based Assays

Cell-based assays are useful for understanding cellular pathways; characterizing targets and their function; identifying, screening, and optimizing ligands or drugs…

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

Most therapeutic antibodies have a Fab’ region that binds the antigen, and an Fc region which binds to and activates…

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Blogs & Articles Driving robust ADCC and T Cell redirection with KILR CD16 Effector Cells

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Publications Regulatory T cells suppress the formation of potent KLRK1 and IL-7R expressing effector CD8 T cells by limiting IL-2

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Publications The SUMOylation inhibitor subasumstat potentiates rituximab activity by IFN1-dependent macrophage and NK cell stimulation

Small ubiquitin-like modifier (SUMO) is a member of a ubiquitin-like protein superfamily. SUMOylation is a reversible posttranslational modification that has…

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Publications Comparison of four in vitro cytotoxicity assays for assessing the potency of CD3-bispecific antibodies redirecting T cells to kill tumor target cells

Evaluation of cytotoxicity is a critical step in the selection of CD3-bispecific antibodies (BsAbs) for cancer immunotherapy.

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Brochures & Flyers Accelerate Immuno-Oncology Drug Discovery & Development

Robust & Reproducible Cell-Based Assays for Potency Testing

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Brochures & Flyers Measure Direct Cytotoxic Cell Death in Co-Culture

Robust, Ready-to-Use ADCC & ADCP Bioassays to Accelerate Your Drug Development Program The KILR® cytotoxicity assay platform developed by Eurofins…

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Brochures & Flyers Eurofins DiscoverX – Delivering Solutions from Discovery to Market

Products & Custom Development Capabilities.

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KILR® Cytotoxicity CD16 Effector Cells

Single donor-derived human primary cells to drive robust & reproducible ADCC & T-cell redirection

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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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Build Your Own Assay Tools

Complete set of parental cell lines, vectors, kits, & retroparticles to build your own stable cell lines & cell-based assays

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Custom Capabilities

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

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