Meeting the challenges of antibody-dependent cellular phagocytosis with more reliable assays. The second of a three-part series.

One tends to think of antibodies as agents of prevention, neutralizing or inactivating viruses in order to block their transmission. The more potent the antibodies, the broader the neutralization tends to be.

But therapeutic antibodies work differently. They exploit a disease’s MO by binding to antigens on the surface of the target cell and then recruiting other arms of the immune system to attack and kill the target. This is precision medicine at its best—no scorched-earth strategy allowed here—so you need to be sure the drug is able to bind properly to the right target.

This is particularly critical in anti-cancer and anti-inflammatory drug development—prime real estate for therapeutic antibodies—where regulators now require Investigational New Drug applications to include functional assays that reflect an antibody product’s mechanism of action (MOA). Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which eliminate target cells in various ways, are two of the more widely studied MOAs. But another potentially useful yet overlooked Fc effector MOA is antibody-dependent cellular phagocytosis (ADCP), in which target cells bound by therapeutic antibody are destroyed via monocyte or macrophage-mediated phagocytosis.

In the first of our three-part Eureka series on ADCP, we discussed many of the obstacles to conventional ADCP functional assays. Today, we look at a novel bioassay that addresses a number of those challenges.

A tedious process

The Fc region or tail of the antibody can engage macrophages and other phagocytes (Pac-Men) of the immune system. Receptors on the macrophages or other phagocytic cells bind to antibodies that are attached to target cells including tumor cells. One of those receptors—FcγRII (CD32a) —is thought to be the dominant player in the induction of ADCP by macrophages and therefore measuring ADCP mediated through this receptor is important in determining bioactivity of a mAb and any biosimilars that follow. Unfortunately, measuring FcγRIIa-mediated ADCP using primary cells can be tedious, inconvenient and complicated, which is largely why ADCP has remained a bit player in bioanalysis.

Until now that is. Regulatory authorities are demanding data on the impact of ADCP on antibody-mediated cytoxicity and laboratories are in the hunt for faster, simpler and more cost-effective in vitro tools to make it happen. One of those tools, a reporter-gene assay that uses the H131 variant of FcγRIIa resulted from a 3-year-old partnership between Charles River and Promega to address challenges in bioactivity testing. Promega developed the reporter-gene assay for routine ADCP testing of therapeutic antibodies and Charles River established the primary ADCP assay using flow cytometry readouts for confirmation and comparison purposes.

The reporter assay

In the reporter assay, primary macrophages are replaced with an engineered Jurkat reporter cell line stably expressing FcγRIIa (H131) and NFAT-RE/luc2. As with primary macrophages, the same signaling for ADCP is activated when the FcγRIIa is bound by antibody that is bound to a target cell, meaning that the reporter assay reflects the in vivo molecular pathway for Fc-receptor-mediated phagocytosis via macrophages (see image).

Figure 1
Figure 1. FcγRIIa ADCP Reporter Bioassay

The non-Hodgkin’s lymphoma drug Rituximab, an early mAb known to induce ADCP activity through FcγRIIa, offers a good test case for how the reporter-gene assay can be used to measure antibody binding. The antibody works by binding to the protein CD20 antigen on the surface of mature B cells and B-cell tumors, then recruits other soldiers of the immune system to kill malignant and normal mature B cells.

In order to be able to assess ADCP function in the reporter gene assay, target cells are first incubated with a titration of Rituximab. Once the therapeutic antibody is bound to the receptor on the target cell surface, the engineered effector cells are added. ADCP pathway activation ensues and results in production of luciferase through activation of the reporter gene NFAT-RE/luc2. Luciferase activity is measured after a 4-24 hour induction period, after addition of the luciferase assay reagent. The dose-dependent response in the microtiter-plate-based assay is then used to quantify the biological activity of the therapeutic antibody. Free antibody does not induce any phagocytosis response, either in the patient or in the in vitro assay. The engineered Jurkat effector cells made in a thaw-and-use format are convenient and help reduce assay run-to-run variability.

From a purely logistical standpoint, the bioassay sidesteps a number of obstacles that one encounters with more classic assays. It eliminates the need to source, purify, do multi-day cytokine differentiation of macrophage effector cells and it avoids the variability of different primary cell donors. The thaw-and-use engineered effector cell method allows same-day results without culturing or having to worry about cell bank concerns. And the test is simple.

And it also performed well. Validation studies found the ADCP reporter bioassay captured differences in bioactivity between the brand name Rituximab—a strong inducer of ADCP—and third generation follow-on molecules of Rituximab. The bioassay was also highly specific. It detected responses when antibody binding occurred and none when non-relevant isotype antibody couldn’t bind to a target cell due to the fact that the receptor was not present on the cell surface. The bioassay was able to quantify the ADCP induction of a variety of drug and research antibodies, and it was stability-indicating in detecting loss of biological activity of Rituximab in this MOA under heat-stress conditions. Ability of bioassays to detect loss of biological activity is important for therapeutic antibodies that are prepared, formulated, stored and transported before being administered.

The primary assay

Unfortunately, efforts to develop the primary ADCP assay were not as fruitful. Because ADCP is a two-step process—the macrophages need to first recognize and bind to the therapeutic antibody bound to the target cell before gobbling it up—using flow-cytometry turned out to be more difficult than expected. Using constitutively stained target cells makes it difficult to distinguish between binding and phagocytosis because the signals in the flow cytometer remain the same for both. Moreover, the dynamic range of the assay is quite small, even for therapeutic antibodies known to be strong inducers of ADCP.

At the end of the day, attempts to optimize the reproducibility and dynamic range of the assay in accordance with Good Manufacturing Practice requirements fell short, confirming why descriptions of ADCP function in the scientific literature are so sparse.

Primary ADCP assays are suitable for MOA confirmation purposes on a nonGMP level, but not appropriate to determine a relative bioactivity on a routine basis or perform biocomparability studies between innovator and follow-on biologics.

So where is the field heading? Will ADCP continue to hover on the sidelines? In the third and final installment of this series, we’ll discuss how the growth of biosimilars and biobetters are driving regulatory authorities to seek a more complete picture of antibody performance. Stay tuned.

How to cite:

Herbrand U., Surowy T., A Reporter on the Trail. Eureka blog. May 26, 2015. Available: