KILR ADCC bioassay kits have been developed for the most-studied tumor models including Raji, Daudi and NCI-N87, and qualified with relevant innovator biologic drugs. These bioassay kits include all the reagents to run an ADCC assay with different immune effector cells and a simple protocol that delivers the results within 1 day.
KILR Bioassay Kits
Effector cells such as PBMCs, NK cells, or KILR CD16 Effector Cells are added to the plated target cells expressing the receptor antigen, which have been engineered to stably express a housekeeping protein that is tagged with enhanced ProLabel® (ePL), a smaller β-galactosidase (β-gal) enzyme fragment using the KILR Retroparticles. When the stable target cell line is used in a cytotoxicity assay, and its membrane is compromised due to cell death, the cells will release the tagged reporter protein into the media. The assay can then detect this reporter protein by the addition of detection reagents containing the larger enzyme acceptor (EA) fragment of the β-gal enzyme. This leads to the formation of the active β-gal enzyme, which hydrolyzes the substrate to give a chemiluminescent output, detected on any bench top luminescence reader.
In healthy cells (left image) with immune effector cells, chemiluminescence is not detected as the reporter protein does not leak out through an intact cell membrane into the media. Alternatively, in cancer cells that are killed by the KILR CD16 Effector Cells, chemiluminescence can be detected directly since cells release the reporter protein into the media and this chemiluminescent signal is proportional to the number of dead cells. Death of any other cell type, including the KILR CD16 Effector Cells present within the co-culture will not affect the assay output, giving the KILR Cytotoxicity assay an unparalleled specificity to detect target cell death within a co-culture system.
KILR Bioassay Assays Applications
- Antibody Dependent Cell-Mediated Cytotoxicity (ADCC) (View Poster)
- Antibody Dependent Cellular Phagocytosis (ADCP)
- Complement Dependent Cytotoxicity (CDC)
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
During development of antibody-based biologics, evaluation of antibody (Fc) effector functions is required by regulators, especially for ADCC. Increasingly regulators are requiring that ADCC assays, especially those used for lot release applications, measure immune cell-mediated killing rather than a surrogate endpoint for antibody engagement of antigen on target cells. KILR ready-to-use bioassays specifically measure killing of antigen-expressing target cells in a co-culture with immune cells, in an easy-to-use, dye-free, and radioactivity-free assay. Based on the industry-validated Enzyme Fragment Complementation (EFC) technology, the KILR ready-to-use bioassays can be applied to multiple immune cell-mediated killing events such as ADCC.
In the KILR bioassays for ADCC application, the target cells expressing the relevant antigen are engineered to stably express a housekeeping protein that is tagged with with a reporter fragment called enhanced ProLabel® (ePL). This reporter fragment is the small enzyme donor fragment of β-galactosidase (β-gal) that is inactive when not paired with its larger enzyme acceptor (EA) fragment. When the stable target cell line is incubated with appropriate effector cells and a test antibody, effector-mediated killing releases the tagged protein into the media. The ePL-tagged reporter protein (also called the KILR Reporter Protein) is detected in the media by the addition of detection reagents containing the EA fragment of β-gal. This leads to the formation of the active β-gal enzyme, which hydrolyzes the substrate to give a chemiluminescent output detected on any bench top luminometer.
Evaluate Diverse Cancer Models with KILR ADCC Assay
The KILR platform is suitable for the detection of ADCC mediated by antibodies targeting multiple antigens in diverse cancer models (stable pools expressing the KILR reporter, to maintain heterogeneity of the native cell line), with different effector types. A. Rituximab-mediated ADCC in four different CD20+ B-lymphoblast KILR models (ARH-77, Daudi, Ramos and WIL2-S) using primary PBMCs at an effector-to-target ratio (E:T) of 25:1. B. ADCC mediated by the anti-CD38 therapeutic antibody, Daratumumab, in the KILR Raji cell model using engineered effector cells (KILR CD16 effector cells) at an E:T of 10:1. 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 at an E:T of 40:1. D. ADCC mediated by the anti-HER2 therapeutic antibody, Trastuzumab (approved for treatment of metastatic breast cancer), in the KILR SKBR3 cell model using primary human PBMCs at an E:T of 25:1. E. ADCC mediated by the anti-EGFR therapeutic antibody, Cetuximab (approved for treatment of metastatic colorectal cancer), in the KILR NCI-N87 cell model using primary human PBMCs at an E:T of 25:1.
Obtain Highly ADCC Reproducibility
High repeatability of ADDC using KILR Raji Bioassay target cells. Dose response of the anti-CD20 antibody rituximab was evaluated by a single analyst on 3 different days using KILR CD16 Effector Cells (E:T = 10:1). Each day represents an independent vial of the target bioassay cells.
Antibody-Dependent Cell-Mediated Phagocytosis (ADCP)
A common human immunoglobulin isotype used for therapeutic intervention is IgG1 that contains two antigen-binding Fab arms and an Fc region. The Fc domain of IgG1 can bind to Fcγ receptors expressed on immune effector cells and mediate target cell death by various mechanisms such as ADCP. Regulatory authorities now commonly require data on the impact of each of this Fc-mediated effector mechanisms for the submitted antibody therapeutic. Getting a reproducible true measure of ADCP, in particular, has been quite challenging. ADCP is a physiologically important MOA of therapeutic antibodies that can be mediated by various immune effector cells, namely: monocytes, macrophages, dendritic cells, and neutrophils through multiple FcγRs. The Fab region of the antibody binds to a specific antigen on the surface of target cells. In contrast, the Fc region of the antibody binds and activates various Fcγ receptors on immune effector cells. Activation of specific Fcγ receptors, such as FcγRIIa, FcγRI, and FcγRIIIa, leads to the continued activation of a complex pathway that triggers phagocytosis and destruction of the target cells within the lysosomes of different effector cells.
In the KILR bioassays for ADCP application, the KILR reporter protein is stably expressed in target cells. When these target cells undergo phagocytosis, the effector cells destroy the target cells including the KILR reporter protein inside the target cells. The amount of total KILR reporter protein in each well is measured through an enzymatic assay with a chemiluminescent output. Lower quantities of KILR reporter protein compared to controls indicate ADCP activity. The workflow schematic below demonstrates the simplicity of the KILR ADCP assay and the scheme of steps followed to measure cytotoxicity using the KILR platform. The schematic uses macrophages differentiated from monocytes (noting other immune effector cells can also be used).
Complement-Dependent Cytotoxicity (CDC)
In the KILR bioassays for CDC application, target cells expressing the relevant antigen are engineered to stably express a housekeeping protein that is tagged with the enhanced ProLabel β-gal reporter fragment. When the stable target cell line is incubated with appropriate effector cells and the test antibody, antibody-coated target cells recruit and activate components of the complement cascade, leading to the formation of a Membrane Attack Complex (MAC) on the cell surface and subsequent cell lysis. The effector-mediated killing releases the tagged protein into the media from target cells. We can detect this KILR Reporter Protein in the media by the addition of detection reagents containing the EA fragment of the β-gal reporter. This leads to the formation of the active β-gal enzyme, which hydrolyzes the substrate to give a chemiluminescent output, detected on any bench top luminometer.