The iLite technology, based on a reporter gene assay format, provides a seamless solution for applications extending across the whole drug development process, as well as for monitoring of biological drugs.
The technology can be developed for virtually any pharmaceutical target and allows an easy, rapid and accurate test format for measurement and quantification of drug activity and immunogenicity. The cells are supplied in an Assay Ready Format, removing the need for cell-culturing and allowing a seamless transfer of the assay between departments or to third party collaborators.
By combining unique features such as a normalization readout and chimeric transcription factors with a highly flexible product format, the iLite technology helps you make the most of your bioassay.
The iLite cell-based assays are based on a simple reporter gene technology. Receptors, specific for a certain target or ligand, are expressed on the surface of a cell. Once the ligand binds to the receptor, this will trigger an intra-cellular signaling cascade, which leads down to a promoter region, fused to the reporter gene, in this case the Firefly Luciferase.
Activation of the reporter gene along with the addition of a substrate will generate light. The amount of light will be correlated to the amount and activity of ligand that was bound to the receptors.
When a ligand binds to its receptor, a specific intracellular signaling pathway is activated, triggering the transcription of a specific reporter gene construct coding for luciferase. When a substrate is added, the luciferase generates light, and the amount of luciferase and thereby the amount and activity of the ligand can then be measured as light emission using a luminometer.
A unique feature of the iLite cells is that they also contain a second reporter gene which is under the control of a constitutive promoter, meaning that it is expressed continuously in any living cell. In other words, the amount of light generated from this second reporter gene will be correlated to the number of living cells and can therefore be used for normalizing results to compensate for differences in cell number.
Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune response where Fc receptor-bearing effector cells recognize and kill antibody-coated target cells that express tumor or pathogen-specific antigens on their surface. Many antibody-based therapeutics rely, at least in part, on their ability to induce ADCC in patients. The iLite ADCC product line offers a convenient and powerful way of measuring the efficacy of antibodies to elicit ADCC in vitro.
The ADCC process is triggered when the effector cell interacts with a drug antibody bound to its target cell. An Fcγllla (CD16a) receptor on the surface of the effector cell binds to the Fc region of the antibody, thus creating a bridge between the effector and target cells.
Following formation of this bridge, the engineered effector cells will, instead of lysing the target cells as in the in vivo situation, produce luciferase though an intracellular pathway and generate luminescence exclusively from this cross-linking and signaling. The strength of the luminescence correlates to the ability of the drug to induce ADCC.
Upon binding to the antibody, NFAT, CREB, NfκB, API and Jak-STAT pathways are activated in the effector cell. The promoter controlling the firefly luciferase gene has been cleverly engineered to contain binding sites for all these pathways. This more closely reflects the in vivo FcγRIIIA signal transduction pathway than commercially available reporter genes that rely solely on the NFAT-responsive promoter.
As most iLite cell lines, the iLite ADCC Effector Cells also have a secondary luciferase readout, from a luciferase gene expressed under the control of a constitutive promotor. This enables normalization of each individual readout according to cell number and compensates for potential matrix effects.
The iLite ADCC system comes with a suite of engineered homologues (+) and (-) target cells that allows differences in ADCC activity to be determined with precision and specificity. It also enables comparisons of ADCC activity between variants therapeutic antibodies that target the same antigen.
Target (+) cells have been engineered to overexpress a specific antigen, either by overactivation of the endogenous expression or by insertion of the gene encoding this antigen.
This gives a stable and controlled expression of the antigen on the cell surface.
Target (-) cells are based on the same cell background as the Target (+) cells but do not present the specific surface target molecule. Hence, the crosslinking between the effector and target cell via an antibody cannot be established and no luminescence from Firefly Luciferase is generated.
Therefore, the specific antigen-negative target cells provide a way of detecting and correcting nonspecific effects sometimes observed in human serum samples.