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Target Cell Killing Bioassays

Targeted killing of tumor or infected cells by effector cells is the primary mechanism of action for many immunotherapy drugs. Thus, it is necessary to demonstrate robust and specific killing of target cells during drug development. Traditional methods used to measure cytotoxicity are not able to distinguish between target and effector cell death. In addition, assays developed to specifically measure the killing of target cells often require radioactive or fluorescent cell labeling, laborious workflows and specialized instruments.

HiBiT Target Cell Killing Platform

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 homogenous. Upon target cell lysis, the assay produces a bright luminescent signal that can be measured using a standard luminometer.

  • Simple, fast, and sensitive technology to specifically measure killing of target cells
  • Flexible platform to measure the activity of a variety of biologic drugs
  • Off-the-shelf HiBiT Target Cells expressing common immunotherapy targets (CD19, CD20, BCMA, and more)
  • Custom development of HiBiT Target Cells and assay optimization to meet your needs
hiBiT TCK assay principle

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 Line Background Drug Targets Potential Applications Test Drugs Available*
A549 Lung carcinoma EGFR mAb, Bispecific, CAR-T cetuximab, trastuzumab, daratumumab (ADCC)
BCMA K562 Chronic myelogenous leukemia BCMA ADC, Bispecific, CAR-T daratumumab (ADCC)
CD3xBCMA (TDCC)
CD19 K562 Chronic myelogenous leukemia CD19 mAb, Bispecific, CAR-T blinatumomab (TDCC)
CIITA K562 Chronic myelogenous leukemia N/A mAb, Bispecific, CAR-T blinatumomab (TDCC)
Claudin 18.2 CHO-K1 Chinese hamster ovarian Claudin 18.2 mAb, Bispecific, CAR-T zolbetuximab (ADCC)
H929 Multiple myeloma BCMA, CD38 ADC, Bispecific, CAR-T daratumumab (ADCC) 
CD3xBCMA (TDCC)
K562 Chronic myelogenous leukemia N/A (Parental) mAb, Bispecific, CAR-T
Membrane TNFα CHO-K1 Chinese hamster ovarian TNFα mAb adalimumab, golimumab, certolizumab (ADCC)
OVCAR3 Ovarian carcinoma MSLN, 5T4, WT, HER2 mAb, Bispecific, CAR-T ertumaxomab (TDCC)
Raji (also CD19-, CD20- & CD19-/CD20- control cell lines) B lymphoma CD19, CD20, CD22, CD38 mAb, ADC,  Bispecific, CAR-T rituximab, daratumumab (ADCC)
blinatumomab (TDCC)
Ramos (also CD19- control cell line) B lymphoma CD19, CD20, CD22, CD38 mAb, ADC, Bispecific, CAR-T rituximab, daratumumab (ADCC)
blinatumomab (TDCC)
SARS-CoV-2-S CHO-K1 Chinese hamster ovarian SARS-CoV-2 S Protein mAb, Bispecific, CAR-T
SK-BR-3 Breast cancer HER2, EpCAM  mAb, Bispecific, CAR-T trastuzumab (ADCC)
SKOV3 Ovarian carcinoma MSLN, 5T4, MUC16, HER2, PD-L1 mAb, ADC, Bispecific, CAR-T trastuzumab (ADCC)
ertumaxomab (TDCC)
T2 Lymphoma HLA-A2+ for TCR-T TCR-T
U937 Lymphoma CD33, CLL-1 mAb, Bispecific, CAR-T gemtuzumab (ADC)

*Drugs are commercially available for the following targets: blinatumomab: CD3xCD19 BiTE; rituximab: anti-CD20; daratumumab: anti-CD38; cetuximab: anti-EGFR; trastuzumab: anti-HER2.

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

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.

Interested in more information about TCK bioassays?

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