RSV’s Multi-Tasking Biologic
How reporter bioassays uncover additional functions for the RSV antibody and why this is important for biosimilars
Respiratory syncytial virus (RSV) is the main reason US children under five end up in the hospital. For children born too early or those with weak immune systems, the virus can cause severe lung inflammation and pneumonia.
Each year, on average, RSV leads to 57,527 hospitalizations and 2.1 million outpatient visits among children younger than 5 years. Globally, RSV infections are estimated to cause more than 250,000 deaths each year.
There are no approved preventive vaccines for RSV, but intramuscular injections of the monoclonal antibody (mAB) palivizumab, which targets an epitope on RSV, can be given passively to high-risk infants and young children during RSV season to ward off infections or serious complications.
Palivizumab, sold under the tradename Synagis, went off patent in 2015 and there are now attempts to develop biosimilars of the drug. In the process, we are learning more about how palivizumab functions, which in turn could make a difference in the development of follow-on biologics.
Palivizumab was brought to market as a passive immunization product that was designed to work pre-emptively in vulnerable children exposed to the virus by neutralizing it. The antibody works by binding to a protein on the surface of RSV that allows the virus to fuse with the cells lining the respiratory tract. This binding process disables the RSV "F" (for fusion) protein and prevents healthy respiratory cells from invasion and infection by the virus. Scientists analyzed the function of the antibody by visualizing the killing of RSV cells in the small wells of a 96-well plate and then measuring the protective effect of the presence of anti-viral antibodies.
Even though neutralization is the proposed mechanism of action (MOA) of palivizumab there is a general requirement to check for Fc effector function for every therapeutic antibody, and data for the Fc effector function capabilities are required for new therapeutic antibodies as well as for biosimilars during development.
Important antibody effector functions like activating antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are mediated via specific receptors on the Fc part of therapeutic antibodies.
According to this perspective in Nature Reviews Drug Discovery, palivizumab belongs to the group of therapeutic antibodies with low Fc effector function. Nevertheless it turned out that palivizumab has abilities beyond neutralization. The mAb can also activate ADCC and ADCP. In case of ADCC, the tips of the Y-shaped antibody binds to an RSV-infected cell and the other end must then bind to other immune cells, so-called natural killer cells, which can then kill the cell and prevent it from replicating. ADCP relies on macrophages to devour the target cell following antibody binding.
Setting up ADCC and ADCP assays with primary cells in the context of therapeutic antibodies that target RSV is a challenge since in addition to the appropriate effector cells like natural killer cells from healthy donors, RSV-infected target cells are required to establish a model system in vitro. The infection of the target cells has an impact on cell viability and thereby on the assay background.
A straightforward way to address these mechanisms of actions (MOAs) in vitro are reporter-gene bioassays. With reporter-based assays we are able to show that palivizumab can induce a significant response to both ADCC via FcgRIII and ADCP via FcγRII (CD32a)—thought to be the dominant player in the induction of ADCP by macrophages. The bioassays included a human lung carcinoma cell line with epithelial-like morphology as target cell line that was infected with a prototype strain of RSV used in the palivizumab antibody.
Reporter-gene 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. This option is allowing us to see a different side of this monoclonal antibody that we need to be aware of.
How might this impact the future development of biosimilars?
Having methods on hand that allow us to do efficient and reliable testing of the Fc effector function potential, even for those therapeutic mAbs with tricky targets, will speed up the understanding of potential MOAs of new therapeutic mAbs based on their in vitro MOA pattern as well as the assessment of biosimilarity.