HiBiT Target Cell Killing Bioassay

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Simple, Fast, Sensitive Technology to Specifically Measure Target Cell Killing

  • Flexible platform to measure activity of a variety of biologic drugs
  • Off-the-shelf HiBiT Target Cells expressing common immunotherapy targets (CD19, CD20, BCMA, etc.)
  • Custom development of HiBiT Target Cells and assay optimization to meet your needs

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HiBiT Target Cell Killing Bioassay

HiBiT Target Cell Killing Bioassay

The HiBiT Target Cell Killing (TCK) Bioassay platform enables highly sensitive and specific measurement of target cell killing induced by a variety of biologic drugs, including monoclonal antibodies (mAbs), bispecific antibodies and CAR-T cells. The assay is simple and homogeneous. Upon target cell lysis, the assay produces a bright luminescent signal that can be measured using a standard luminometer.

Assay Principle

tck-assay-schematic

Principle of the HiBiT TCK Bioassay. Cytotoxic mAbs and/or effector cells are incubated with target cells expressing a HiBiT fusion protein. Upon killing of the target cell, the HiBiT fusion protein is released and binds extracellular LgBiT to create a functional NanoBiT® Luciferase enzyme. Luminescence is measured using a luciferase substrate and the GloMax® Discover System.


Available HiBiT Target Cells

Target Cells* Cell Type Select Target Expression Example Drug Products
Endogenously Expressed Targets
Raji Cells (HiBiT) B Cell Lymphoma CD19, CD20, CD22, CD38 rituximab (anti-CD20 mAb), blinatumomab (CD19xCD3 bispecific Ab), daratumumab (anti-CD38 mAb), Yescarta, Kymriah (anti-CD19 CAR-T cells)
Raji CD19-KO Cells (HiBiT) CD20, CD22, CD38
Raji CD20-KO Cells (HiBiT) CD19, CD22, CD38
Raji CD19/CD20-KO Cells (HiBiT) CD22, CD38
Ramos Cells (HiBiT) B Cell Lymphoma CD19, CD20, CD22, CD38 rituximab (anti-CD20 mAb), blinatumomab (CD19xCD3 bispecific Ab), daratumumab (anti-CD38 mAb), Yescarta, Kymriah (anti-CD19 CAR-T cells)
Ramos CD19-KO Cells (HiBiT) CD20, CD22, CD38
H929 Cells (HiBiT)  Multiple Myeloma BCMA ciltacabtagene autoleucel (anti-BCMA CAR-T cells), belantamab mafodotin - blmf (anti-BCMA ADC), idecabtagene vicleucel (anti-BCMA CAR-T cells), BCMAxCD3 bispecific Ab (in development)
OVCAR3 Cells (HiBiT) Ovarian Carcinoma MSLN, 5T4, WT, HER2 ertumaxomab (HER2xCD3 bispecific Ab), trastuzumab (anti-HER2 mAb)
SKOV3 Cells (HiBiT) Ovarian Carcinoma MSLN, 5T4, MUC16, HER2, PD-L1 ertumaxomab (HER2xCD3 bispecific Ab), trastuzumab (anti-HER2 mAb)
SK-BR-3 (HiBiT) Breast Adenocarcinoma HER2, EpCAM trastuzumab (anti-HER2 mAb)
A549 Cells (HiBiT) Lung Carcinoma EGFR cetuximab (anti-EGFR mAb)
U937 Cells (HiBiT) Lymphoma CD33, CLL-1 gemtuzumab ozogamicin (anti-CD33 ADC)
T2 Cells (HiBiT) Lymphoma HLA-A2+ Cancer vaccine applications
Exogenously Expressed Targets
K562 Cells (HiBiT) Chronic Myelogenous Leukemia N/A N/A
CD19 K562 Cells (HiBiT) CD19 blinatumomab (CD19xCD3 bispecific Ab), Kymriah (anti-CD19 CAR-T cells)
BCMA K562 Cells (HiBiT) BCMA ciltacabtagene autoleucel (anti-BCMA CAR-T cells), belantamab mafodotin - blmf (anti-BCMA ADC), idecabtagene vicleucel (anti-BCMA CAR-T cells), BCMAxCD3 bispecific Ab (in development)
Claudin 18.2 CHO-K1 Cells (HiBiT) Claudin 18.2 mAb and CAR-T cell therapies (in development)
CIITA K562 Cells (HiBiT) Various Cancer vaccine applications
Membrane TNFα CHO-K1 Cells (HiBiT) Chinese Hamster Ovary Membrane TNFα adalimumab, certolizumab, golimumab (anti-TNFα mAb)
SARS-CoV-2 S Protein CHO-K1 Cells (HiBiT) SARS-CoV-2 S Protein anti-SARS-CoV-2 mAb (in development)

*HiBiT Target Cells are available in CPM and Thaw-and-Use formats.

 

Don't see a target cell that you need? Contact our Assay Development and Services team about custom assay development.

Applications

The HiBiT TCK platform can be used to measure TCK activity induced by a variety of biologic drug modalities. The data included here are representative of three applications: CAR-T cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), and T cell-dependent cell-mediated cytotoxicity (TDCC).

CAR-T Cell-Mediated Cytotoxicity

Antigen-specificity of NanoBiT™ TCK Assay
Antigen-specificity of NanoBiT™ TCK Assay

Extended assay incubation time reveals serial TCK activity. T cells transduced with CAR-19 or a GFP control lentivirus were combined with HiBiT Target cells (Ramos) at different effector:target (E:T) ratios. After incubation for 24 hours (Panel A) or 48 hours (Panel B), NanoBiT® Extracellular Detection Reagent was added, and luminescence was read on a GloMax® Discover plate reader. The EC50 shifts left over time, indicating serial TCK at lower E:T ratios.


Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

PBMC-mediated TCK assay
ADCC activity assay with SARS-CoV-2

TCK activity measured using the PBMC ADCC Bioassay. Panel A. PBMC-mediated killing of Raji (HiBiT) Target Cells by anticancer therapeutic mAb, rituximab. Panel B. PBMC-mediated killing of SARS-CoV-2 S CHO-K1 (HiBiT) target cells by an anti-SARS-CoV-2 spike (S2) antibody.


T Cell-Dependent Cell-Mediated Cytotoxicity (TDCC)

TDCC bioassay
TDCC bioassay

The TDCC Assay measures target cell-specific killing. Thaw and Use CD8+ T Cells (TDCC Qualified) were combined with HiBiT-expressing target cells and appropriate Bispecific T Cell Engagers (BiTEs.) After 18–24 hours incubation, NanoBiT® Extracellular Detection Reagent was added, and luminescence was read on a GloMax® Discover plate reader. The assay enables sensitive measurement of bispecific antibody potency in a homogenous format with no medium transfer required.

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Petra

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