As outlined in ICH Q6B “Test Procedures and Acceptance Criteria for Biotechnological/Biological Products” (CPMP/ICH/365/96), the regulatory expectation for any developmental biopharmaceutical is that a wide range of analytical methodologies are used to characterize the therapeutic. Generally, this means the application of orthogonal physicochemical and biophysical techniques to provide a comprehensive understanding of the active molecule. Monoclonal antibodies, ADC and other immunoglobulin derived biopharmaceuticals are, however, a special case. Due to the potential of these molecules to induce immunological responses through the Fc region of the IgG protein, there is an expectation these effector functions will be evaluated to determine whether they have an impact on the product safety and efficacy (EMA, Guideline on Development, Production, Characterization and Specifications for Monoclonal Antibodies and Related Products). Specifically, they require Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity assay (CDC) and possibly also Antibody-Dependent Cellular Phagocytosis (ADCP) to be evaluated. It should be noted that not all antibodies have ADCC, CDC or ADCP activity and indeed they can be engineered to be devoid of such activity. Nevertheless, the expectation is that secondary effector function is evaluated, even if it is only to demonstrate absence of activity. Completing this assessment in the early stages, enables a clear risk assessed approach to be developed to enable a clearer drug development pathway
Initially, in a risk assessed approach, rapid evaluation of the Fcγ receptor and C1q interactions with the therapeutic can be performed in a binding assay, such as surface plasmon resonance or ELISA. However, if binding is confirmed, the secondary effector functions need to be evaluated in a cell based bioassay.
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
ADCC is an innate immune response resulting in lysis of target cells, mediated by antibodies specifically binding to cell surface antigens. When therapeutic antibodies with ADCC activity bind to cells that express the target antigen, they induce non-specific cytotoxic cells, such as Natural Killer (NK) cells, to interact with the Fc region of the antibody via the FcγIIIa receptor. These cytotoxic immune cells are then activated releasing cytokines such as IFN-γ or TNF-α and cytolytic granules that induce cell death.
Several ADCC cell-based assays formats are available for determining the efficacy of antibodies. These require target cells that express the surface antigen as a source of cytotoxic cells. These cytotoxic cells can be a cell line, such as NK-92 or Jurkat cells, or alternatively peripheral blood mononuclear cells (PBMCs) can also be used. Traditionally the assay endpoint was radioactive Cr51 release from pre-loaded target cells; however improved methods measuring LDH release or using fluorescent reporter gene assays are now available.
Complement-Dependent Cytotoxicity assay (CDC)
The complement system is part of the innate immune system comprising a series of inactive plasma proteins. When a therapeutic antibody with CDC activity binds to cells that express the target antigen, it induces binding to the complement C1q protein, which in turn induces a cascade of specific hydrolytic events of other complement proteins. This cascade results in the activation of the membrane attack complex (MAC) on the surface of the target cell. These terminal complement proteins polymerize to form pores in membranes that can kill the targeted cell.
As with the ADCC assay, CDC assay formats require a cell line that expresses the target surface antigen. Plasma represents a convenient source of C1q and other complement proteins, although recombinant complement proteins are also available. The CDC reaction results in cell death, which can be determined using fluorescent or luminescent endpoints.
Antibody-Dependent Cell Phagocytosis (ADCP)
The least common Fc effector function is ADCP. With this system, antibodies bind to cell surface antigens and induce cell death by phagocytosis rather than cell lysis. The Fc region of the antigen bound antibody binds mainly to FcγIIa receptors on effector cells such as monocytes, macrophages, neutrophils or dendritic cells, which in turn induces phagocytosis of the target cell and subsequent cell death.
ADCP assays require a target cell line expressing the surface antigen and also a source of phagocytic cells. Such cells can be derived from PBMCs or alternatively specific cell lines are generally used. The endpoint of the assay can be fluorescent activated cell sorting with immunocytochemistry or ADCP reporter gene assays are also now available producing a fluorescent read out.
Antibody-Induced Effects on Cell Proliferation and Apoptosis
As well as the antibody-mediated mechanisms of cytotoxicity, which are facilitated through the Fc region of the molecule, antibodies can directly inhibit cell proliferation and/or induce apoptosis in cells expressing the target antigen. The mechanism of action by which such antibody-mediated effects can be activated are varied and could include antigen cross-linking, activation of death receptors and inhibition of growth or other signaling pathways. Irrespective of how the effect is induced, the end result is usually perturbation of the cell cycle and/or induction of apoptosis.
As the effects are directly mediated by the antibody, such cell-based assays only require a cell line expressing the target antigen and no effector agent is required, other than the antibody itself. For determination of proliferative effects, colorimetric viability indicators, such as ® or 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) can be used as endpoints. With apoptosis, standard cell death endpoints, such as LDH release, can be used. As LDH release is relatively non-specific of apoptosis, alternative markers include caspase-3, can provide more specificity. It is increasingly common to find that the analysis of cell proliferation and apoptosis/cell death are multiplexed into a single assay; hence comparative data on proliferation effects and apoptosis can be obtained using the same cells.
For antibody based biopharmaceuticals the regulatory expectation is that a characterization package will be compiled to provide a comprehensive understanding of the molecule and its mode of action. As antibodies can have biological activity in addition to the primary effect of binding to their respective antigen, an understanding of the biology is required as a component of the characterization package. Should binding of the therapeutic antibody to Fcγ receptors or C1q be confirmed, then a series of cell-based bioassays are available to provide the necessary information of antibody effector functions.