Bioassays for Checkpoint Inhibitors
Reporter-gene bioassays present a reliable method to determine the responses of immunotherapies
Immune checkpoint therapy, which targets regulatory pathways in T cells to amplify antitumor immune responses, has been a game-changer in the treatment of some cancers. Sparked by the early success of therapeutic monoclonal antibodies (mAbs) which inhibit programmed cell death and CTLA4, over 100 experimental inhibitors are being developed alone or in combination with bispecific mAbs or cytostatic agents such as antibody drug conjugates.
But because the adaptive immune responses from T cells and B cells are so complex and multi-faceted, analyzing a particular biologic’s mechanism of action using primary assays—ones that focuses on a single drug target—can be really difficult. The co-activating signal of the antigen-presenting cell needs to be mimicked to achieve activation and primary T cells are required as effector cells.
Laboratories can use a number of highly relevant primary assays, from measuring T-cell proliferation to the cell death of the target cells, to evaluate their antibodies, but the multiple cell types present in the assay, the variability of the sources of the primary cells and, in some situations, long assay times, make them poor choices for routine testing in a Good Manufacturing Practice (GMP) environment.
So manufacturers are using reporter-gene assays to perform their bioanalysis. Reporter-assays are designed to, well, report things. They work the beat, like any good journalist, and gather information. In the case of therapeutic antibodies, the reporter-gene goes undercover and is placed under the control of the antibody to determine its function.
A reporter-gene bioassay that we have used employs a modified human T lymphocyte cell line as a CTLA4 expressing effector cell line and antigen-presenting cells. The effector cells express luciferase in dependence of the CTLA4 down-regulating pathway and if CTLA4 is blocked, the gene for luciferase—the enzyme responsible for the firefly’s light production—is expressed steadily and can be measured using luminescence plate readers.
A different kind of bioassay, called a blockade bioassay, is being used on PD-1/PD-L1, the checkpoint proteins that Keytruda and Optivo are designed to inhibit. The bioassay consists of PD-1 effector cells and target cells expressing human PD-L1 and an engineered cell surface protein designed to activate cognate T cell receptors (TCR) in an antigen-independent manner. When the two cell types are co-cultured, the PD-1/PD-L1 interaction inhibits T cell receptor signaling and luminescence.
By adding either an anti-PD-1 or anti-PD-L1 antibody that blocks the PD-1/PD-L1 the interaction releases the inhibitory signal and results in TCR activation and luminescence.
Therapeutic mAbs, like those that interfere with T-cell signaling, have the same features as all other therapeutic mAbs, which means that they might be capable to mediate one or more mechanisms of action (MoAs) of the classical immunological pathway group via their Fc regions. For example Ipilimumab can mediate ADCC, which can be shown nicely by using a CTLA-4 expressing target cell line and a surrogate ADCC reporter effector cell line. Reporter assays can be useful in assessing the function of checkpoint inhibitors with regards to induction of classical immunological pathways like ADCC and ADCP.
Surrogate reporter bioassays to measure classical immunological pathways’ mechanisms of action, like ADCC, ADCP and specific reporter bioassays—like anti CTLA4 and PD1—which measure the activity of immune checkpoint therapeutic mAbs with genetically engineered cell lines mimicking the T cell and the antigen presenting cell in a mechanism-of-action reflecting manner are much less variable in format. This allows these assays to be used in GMP bioactivity testing. Surrogate reporter assays also are stability-indicating and suitable for high-throughput biosimilarity assessment and accepted by regulatory authorities.
It’s important to manage expectations no matter which bioassay is used to measure functional responses of therapeutic antibodies. The tests, based on models with primary cells are extremely challenging. All are highly relevant, but can also be problematic due to multiple cell types being present in the assay, long assay performance times and variability with sources of primary cells.