Measuring Direct, Bystander, and Off-Target ADC Killing with the HiBiT TCK Platform

Sponsored content brought to you by

The efficacy of biologic-based immunotherapies relies on their ability to induce apoptosis and cell death through the recruitment and activation of immune effector cells. Biologic-

based immunotherapies come in a variety of formats, such as antibody drug conjugates (ADCs), chimeric antigen receptor (CAR) T cell therapy, and monoclonal antibodies (mAbs), to name a few. For payload-linked formats like ADCs, one challenge is verifying delivery of the cytotoxic payloads to only targeted cells in a heterogeneous tumor environment.

To help address this challenge, Promega’s HiBiT Target Cell Killing (TCK) platform is a streamlined, bioluminescent cell-based system that measures cytotoxicity with specificity, simplicity, and sensitivity during therapeutic development. The platform supports all major cell-killing paradigms, including CAR-T cell-mediated killing, antibody-dependent cellular cytotoxicity (ADCC), T cell-dependent cellular cytotoxicity (TDCC), antibody-dependent cellular phagocytosis (ADCP), and ADC bystander killing.

How HiBiT TCK works

Target cells are engineered to express a HiBiT fusion protein that remains intracellular until cell death. Upon membrane disruption, HiBiT is released into the medium, where it binds cell-impermeable LgBiT to form functional NanoBiT® luciferase. The luminescent signal is proportional to target cell death alone, with no contribution from effector cells, making the platform ideal for co-culture experiments. The workflow requires no washing, loading, or staining steps and produces robust signal-to-noise with as few as 2,000 target cells per well.

A growing library of thaw-and-use, functionally tested cell lines addresses blood cancer targets, including B cell lymphoma and leukemia, myeloid leukemia, and multiple myeloma (Raji, Ramos, H929), as well as solid tumor targets for ovarian carcinoma, breast adenocarcinoma, and lung carcinoma (SKOV3, SK-BR-3, OVCAR3). HiBiT-containing target cell lines can also be custom tailored.

ADCs deliver cytotoxic payloads to antigen-expressing tumor cells, but tumors are antigenically heterogeneous. When an ADC’s payload is released inside a targeted cell, it can diffuse and kill neighboring antigen-negative cells. This bystander killing can extend therapeutic coverage across a mixed tumor, or it can damage healthy adjacent tissue. The outcome depends largely on linker-payload chemistry: Cell-permeable, cleavable payloads produce high bystander activity, while cell-impermeable payloads do not. Characterizing this activity during development is essential but difficult with conventional cytotoxicity assays, which cannot attribute cell death to specific populations in a co-culture.

ADC bystander testing

The Bystander Killing Assay uses a three-cell-line design: wild-type target cells (HiBiT) to measure direct ADC cytotoxicity, wild-type target cells (“Dark”, no HiBiT) to serve as the antigen-expressing bystander driver, and antigen-KO cells (HiBiT) to specifically measure bystander killing. In the assay, ADC binds the “Dark” antigen-positive cell and the payload is internalized. If the released payload diffuses and kills neighboring antigen-KO HiBiT cells, the resulting luminescence is specific to bystander killing. Because HiBiT remains intracellular until membrane disruption, only dead target cells contribute signal. Effector or bystander driver cells do not.

Proof of concept

To demonstrate proof of concept, the HiBiT TCK bioassay was used on SKOV3 cells with two approved HER2-targeting ADCs, disitamab vedotin with a cleavable MMAE payload and ado-trastuzumab emtansine with a non-cleavable DM1 payload. The assay confirmed positive bystander killing with disitamab vedotin. Conversely, Kadcyla demonstrated a negative bystander killing effect. CellTiter-Glo, which reports total well viability without distinguishing cell populations, was unable to make this distinction, demonstrating the power of the HiBiT TCK Bioassay. The same principle was validated with loncastuximab tesirine on Raji/CD19-KO co-cultures.

The examples validate that the streamlined HiBiT TCK platform measures cytotoxicity with specificity, simplicity, and sensitivity. Since the resulting luminescent signal is specific to the engineered target cell, the bioassay is well-suited for mixed co-culture experiments during development efforts. Beyond bystander applications, the HiBiT TCK platform supports ADCC with PBMC effectors, TDCC with CD8+ T cells, and CAR-T killing assays across a 4–72 hour time course.

Learn more www.promega.com.

The post Measuring Direct, Bystander, and Off-Target ADC Killing with the HiBiT TCK Platform appeared first on GEN - Genetic Engineering and Biotechnology News.