KILR CYTOTOXICITY PRODUCT SOLUTIONS

The KILR cytotoxicity assay platform specifically measures the killing of antigen-expressing target cells in co-culture with effector cells. This robust and flexible platform can be used for the quantitation of direct cell death mediated through multiple MOAs such as ADCC, ADCP, CDC, T-cell redirection, and CAR-T.

The KILR ADCC assay response is highly specific. A. Cetuximab mediates an ADCC response in U2OS cells over-expressing EGFR, but not in native U2OS cells that have low levels of endogenous EGFR. B. A highly specific ADCC response is observed with Cetuximab targeting U2OS cells expressing EGFR as opposed to that of Rituximab that targets CD20 that results in no ADCC. NK = Natural Killer. E:T = Effector Cells:Target Cells. S/B = signal-to-background.

KILR ADCC Assays are compatible with multiple effector types. A. ADCC using primary PBMCs from 3 different donors in the KILR SKBR3 model B. ADCC with two HER2-targeting antibodies using primary NK cells in the KILR SKBR3 model. C. ADCC with Trastuzumab using and engineered NK-92 cell line as effector cells, with the NK-92-resistant SKOV3 cell pool. NK = Natural Killer. E:T = Effector Cells:Target Cells. S/B = signal-to-background.

Generate cell-based cytotoxicity assays in any dividing cell type. Using Enzyme Fragment Complementation (EFC), which is based on a split β-galactosidase (β-gal) enzyme you can create your own cytotoxicity assays. Target cells expressing the receptor antigen of choice can be engineered to stably express a protein (housekeeping protein) tagged with the small β-gal enzyme donor (called the enhanced ProLabel (ePL) or β-gal reporter fragment) using KILR Retroparticles. When the stable target cell line is used in an EFC-based cytotoxicity assay (e.g., ADCP, ADCC, CDC), the EFC reaction can be measured with the addition of the large β-gal enzyme acceptor (EA) fragment that creates the active β-gal enzyme. With the addition of hydrolyzing substrate, a chemiluminescent output can be detected on any bench top luminometer.

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 CDC 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 addition of test antibody to target cells. Following opsonization, appropriate complement is added to the wells containing the target cells. Complement-mediated killing releases the KILR reporter protein into the media, which is detected by the addition of detection reagents and results in a chemiluminescent signal that can be read on any standard bench top luminometer.

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, 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.

The KILR CDC assay principle. CDC is mediated by the C1 complex that initiates deposition of C3b fragments on target cells. The C3b in conjunction with C4b and C2a forms the C5 convertase enzyme that cleaves C5 into C5a and C5b. C5b then participates in the formation of the membrane attack complex or MAC (C5b-C6-C7-C8-C9). MAC is responsible for the cytolytic function of the complement system resulting in subsequent target cell lysis. The KILR reporter protein (including the β-gal ED fragment also called ePL) is stably expressed in the target cells and released into the media when these target cells undergo lysis as a result of MAC formation. The amount of total KILR reporter protein is detected by the addition of detection reagents containing the β-gal EA fragment. Complementation of ED and EA facilitates detection of the remaining amount of KILR protein present, and with the addition of subtrate, hydrolysis occurs and a chemiluminescent signal can be detected on any bench top luminometer.

The KILR ADCC assay application. A. Graphic illustration of the KILR ADCC MOA. B. Rituximab-mediated ADCC in four different CD20+ B-lymphoblast KILR models (ARH-77, Daudi, Ramos, and WIL2-S) using primary PBMCs. C. ADCC mediated by the anti-CD38 therapeutic antibody, Daratumumab, in the KILR Raji cell model using engineered effector cells (KILR CD16 effector cells). D. 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. This data demonstrates that KILR ADCC assays can be used to evaluate diverse cancer models. E:T = Effector Cells:Target Cells.

The KILR ADCP assay application. A. Graphic illustration of the KILR ADCP MOA. B. Robust ADCP observed with trastuzumab with KILR NCI-N87 cell pool (a HER2+ solid tumor model). C. A robust dose-dependent increase in % ADCP was observed with increasing concentrations of rituximab. An E_MAX value of 83% was obtained with a low EC₅₀ of 2.4 ng/mL. E:T = Effector Cells:Target Cells.

The KILR CDC assay application. A. Graphic illustration of the KILR CDC MOA. B. CDC activity was assessed with KILR Raji bioassay cells. This assay responds specifically to rituximab and exhibits a wide dynamic range as indicated by the % lysis of the KILR target cells.

The KILR T-cell redirection assay application. A. Graphic illustration of the KILR 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.