KILR CYTOTOXICITY CD16 EFFECTOR CELLS

KILR ADCC Assay Workflow using KILR CD16 Effector Cells

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 cytotoxicity platform is based on the industry-validated Enzyme-Fragment Complementation (EFC) technology. EFC is based on two recombinant β-galactosidase (β-gal) enzyme fragments that act as an enzyme acceptor (EA) and an enzyme donor (ED). Separately, the fragments are inactive, but when combined, they form an active β-gal enzyme that hydrolyzes its substrate to produce a chemiluminescence signal.

KILR ADCC Assay Principle using KILR CD16 Effector Cells

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. Overall, the single donor-derived KILR CD 16 Effector Cells are introduced to evaluate ADCC, and these effector cells are universal and can be used in any ADCC assay or T-cell redirection applications.

Obtain Robust ADCC Assay Results with Single-Donor Derived KILR CD16 Effector Cells

Improved ADCC assay performance with KILR CD16 Effector Cells. Single-donor derived KILR CD16 Effector Cells are engineered cytotoxic T lymphocytes (CTLs) derived from a single donor that are transfected with CD16 (FcγRIIIa- V158). Robust ADCC was observed with KILR CD16 effector cells than with primary cells. In KILR Raji cells treated with Rituximab, KILR CD16 Effector Cells mediate a 2.5-fold higher assay window and E_Max than primary natural killer (NK) cells when used at the same effector-to-target (E:T) ratio (E:T = 10:1). The difference in assay window and E_Max is even more pronounced when comparing primary PBMCs (used at an E:T = 25:1).

Achieve Significantly Larger Assay Windows to Better Analyze Antibody Activity

Large assay windows with KILR CD16 Effector Cells compared to PMBCs. A. KILR cytotoxicity model ARH77 (CD20+) was opsonized with rituximab and then incubated with either KILR CD16 Effector Cells (E:T = 10:1) or PBMCs (E:T = 25:1). B. KILR cytotoxicity model SKOV-3 (HER2+) was opsonized with trastuzumab and then incubated with KILR CD16 Effector Cells (E:T = 12.5:1) or PBMCs (E:T = 25:1). After addition of KILR detection reagents in both assays, a 4- or 16-fold larger assay window was observed in the ARH77 or SKOV3 models, respectively, with KILR CD16 Effector Cells relative to PBMCs.

Detect Differences in Glycan Profiles for ADCC Assays

Infliximab comparability studies showing KILR CD16 Effector Cells are able to detect glycan profiles in an ADCC assay. A. Glycosylation profile of the innovator and biosimilar infliximab. The glycan profile of both molecules are consistent with a monoclonal antibody that has been expressed in mammalian cell lines. Most peaks in the biosimilar chromatogram are also observed in the innovator chromatograms although there are some variations. There are higher levels of some sialic acid species and an increase in galactosylation of G1F and G2F, and there is decreased afucosylation in the biosimilar profile that can have an impact on ADCC activity. B. ADCC activity of infliximab was subsequently assessed using purified NK cells from V/V and F/F donors, and compared with KILR CD16 Effector Cells. The following potencies were returned for the biosimilar molecule during the assay: V/V donor – 29.7%, F/F donor – 38.7%, KILR CD16 cells – 41.0%. This data is part of a comparability study conducted by BioOutsource Sartorius Stedim.

T-Cell Redirection Application using Bi-specific T-cell Engagers (BiTEs)

The KILR T-cell redirection assay application. A. Graphic illustration of theKILR t-cell redirection MOA. BiTEs are chimeric antibodies with dual specificities that target tumor cells by harnessing the power of the immune effector cells. The BiTE molecule binds to a tumor-associated antigen such as CD19. The KILR CD16 Effector Cells, which express CD3 endogenously, rapidly kills target cells via t-cell redirection. B. KILR CD16 Effector Cells were incubated with KILR Raji cells and blinatumomab (an FDA approved BiTE molecule) for 4, 5, or 6 hours at 37°C. Optimal assay window (max killing of >60%) was observed at 6 hour incubation with KILR CD16 effectors with an EC₅₀ of 15.5 pg/mL.