Method for Measurement of Antibody-Dependent Cellular Phagocytosis.

Antibody-based therapeutics are powerful tools to treat disease. While their mechanism of action (MOA) always involves binding to a specific target via the antibody-binding fragment (Fab) region of the antibody, the induction of immune-mediated effector functions through the fragment crystallizable (Fc) region is a vital aspect of antibody therapeutics targeting tumor cells. Cross-linking of the Fc gamma receptors (FcγRs) via cell-bound antibodies activate immune effector cells, leading to antibody-dependent cellular cytotoxicity via natural killer (NK) cells. Linking of FcγRs on macrophages triggers the process of antibody-dependent cellular phagocytosis (ADCP), where antibody-opsonized target cells are internalized in phagosomes and degraded through the process of phagosome maturation and acidification. ADCP activity can be challenging to measure accurately due to the difficulty in differentiating target cells that are bound to a macrophage versus those that are internalized within phagosomes. In this chapter, we describe a protocol that measures ADCP activity by labeling target cells with a pH-sensitive dye that fluoresces brightly in mature phagosomes. The ADCP activity of therapeutics is then measured via flow cytometry. This assay is capable of detecting glycosylation differences arising from manufacturing processes and is suitable for evaluation of ADCP activity of monoclonal antibodies (mAb) to support in vitro biological characterization of drug candidates and lead candidate selection for desirable effector functions.

Introducing dendritic cell antibody internalization as an immunogenicity risk assessment tool.

Aim: Immunogenicity risk assessment assays are powerful tools that assess the relative immunogenicity of potential biotherapeutics. We detail here the development of a novel assay that measures the degree of antibody internalization by antigen-presenting cells as a predictor of immunogenicity. Results & methodology: The assay uses the fluorescence signal from the antibody bound to the outside of the cell as well as inside the cell to determine internalization. To calculate the amount of internalized antibody, the fluorescent signal from the outside was subtracted from the fluorescent signal from the inside, which is referred to as the internalization index. Conclusion: This assay format demonstrated that antibody-based biotherapeutics with higher clinical immunogenicity internalized to a higher degree than therapeutic antibodies with lower clinical immunogenicity.

A novel method for determining antibody-dependent cellular phagocytosis.

Antibody-based therapeutics are powerful tools to treat disease. While their mechanism of action (MOA) always involves binding to a specific target via the Fab region of the antibody, the induction of effector functions through the Fc region of the antibody is equally important for antibody therapeutics designed to deplete tumor cells. By binding of the Fc region to Fc gamma receptors (FcγRs) on the surface of immune cells or complement factors, antibody therapeutics exert effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), both of which induce target cell death and aid in the efficacy of treatment. Another major Fc effector function is antibody-dependent cellular phagocytosis (ADCP). ADCP is the mechanism by which antibody-opsonized target cells activate the FcγRs on the surface of macrophages to induce phagocytosis, resulting in internalization and degradation of the target cell through phagosome acidification. ADCP has been implicated as a major MOA of several biologics, but this activity is difficult to measure in in vitro. Most assays measure the association of target cells and macrophages; however, co-localization can represent cell attachment rather than internalization. Here, we describe the development of a novel method to accurately measure ADCP activity. By labeling target cells with a pH sensitive dye that only fluoresces in mature phagosomes, the ADCP activity of antibody therapeutics can be accurately quantitated via flow cytometry.

Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier.

Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.