KILR CYTOTOXICITY BIOASSAYS

KILR ADCC bioassays deliver high repeatability and reproducibility. A. A dose response of the anti-CD20 antibody Rituximab was evaluated in the respective target cells by a single analyst on three different days using KILR Raji bioassay target cells and KILR CD16 Effector Cells. The results demonstrate the low variability in EC50, S/B (signal-to-background), and EMAX values over the three days indicating the high repeatability of the ADCC assay. Comparable data was obtained with the KILR Daudi model (data not shown). B. Functional response analysis of KILR ADCC bioassays by two analysts over two days shows excellent concordance in curve shape, S/B, and EC₅₀.

Comparison of rituximab-mediated ADCP in two different assay formats – flow cytometry vs the KILR enzyme fragment complementation (EFC) format. Monocytes from the same healthy donor were isolated and differentiated to M0 macrophages prior to use in each assay. A. Expression of CD11b and CD14 was assessed on the polarized macrophages by flow cytometry at the time of harvest to confirm the differentiation state. B. Flow-based and C. KILR EFC-based assay results normalized to vehicle-treated cells to calculate % ADCP. Overall, a higher percentage of phagocytosis was observed when using the KILR assay, indicating a more effective ADCP assay format. E:T = Effector cells (macrophage):target.

A well-structured process for bioassay development yields assays that are robust and reproducible. The process begins with selecting the assay that most appropriately reflects the MOA of the drug, followed by optimization of assay parameters. Optimization through qualification steps includes refining conditions with the originator drug or reference molecule to establish robustness, precision, accuracy, and linearity of assay.

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.

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.

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 (e.g. KILR CD16 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.

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.