Functional Characterization of Therapeutic Antibodies

Introduction

Monoclonal antibodies (mAbs) and related biological products often present as ideal therapeutics largely due to:

1. Their high specificity and affinity to the target antigen, allowing for a high level of efficacy with fewer adverse outcomes.
2. Their diversity in structure and function, allowing for a wide range of therapeutic applications and targets.
3. Their amenability to modifications through antibody engineering and recombinant production, allowing for the generation of many antibody-based derivatives including chimeric antibodies, humanized antibodies, bispecific antibodies, antibody fragments (ie. Fabs, nanobodies, diabodies, triabodies), antibody-drug conjugates, and antibody-PROTACs.

To meet the acceptance criteria of regulatory authorities (ie. FDA, EMA), rigorous programs are required to provide a complete characterization of therapeutic mAbs and related products. This includes:

1. 1. Structural and physicochemical characterization
   2. Immunochemical characterization
   3. Functional characterization
   4. Purity and contaminant characterization
   5. Quantification

In this article, we will discuss the different mechanisms of action (MOAs) of therapeutic antibodies and the strategies used to assess their functions.

Functions of Therapeutic Antibodies

MAbs are multifunctional by nature and their efficacy is largely attributed to the antigen-binding fragment (Fab) region and the crystallizable fragment (Fc) region. These two regions can define the antibody’s function and MOA within a specific immune response or biological process. Common MOAs (Figure 1) that drive the biological activities of mAb therapeutics and related products include:

1. Blocking of ligand-receptor interactions – mAbs can bind to either side of the ligand-receptor interface to block binding and disrupt the subsequent signal transduction pathway.
2. Effector engagement for ADCC, ADCP, and CDC – mAbs can engage effector functions through binding of the Fc region to the Fc receptors and complement proteins to effectively deplete target cells.
   • ADCC, or antibody-dependent cellular cytotoxicity, and ADCP, or antibody-dependent cellular phagocytosis, are engaged upon binding of the Fab to the antigen-expressing cell, allowing the mAb to recruit effector cells (ie. natural killer (NK) cells, macrophages) that express Fc surface receptors, which bind to Fc, resulting in the elimination of the mAb-labeled antigen-expressing cell via lysis or phagocytosis.
   • CDC, or complement-dependent cytotoxicity, is engaged upon binding of the C1 complex to the antibody-antigen complex, activating a cascade of complement proteins to form an immune complex that is eliminated by effector cells.
   • Cytotoxic T cells are engaged through bispecific mAbs to direct the T cells towards tumour cells by binding to both the T cell receptors and the antigen-expressing tumour cell, leading to T cell proliferation and the release of cytotoxic factors (ie. IL-2, INFy).
3. Signaling downregulation by receptor internalization and degradation – mAbs binding to nonoverlapping epitopes can be combined synergistically for enhanced internalization and downregulation of epidermal growth factor (EGF) receptors to effectively shut down their signaling pathways that are involved in many human cancer types.
4. Targeting by drug delivery – mAbs can be conjugated to cytotoxic payloads (ie. radioisotopes, toxins, small molecules, or cytokines) and delivered directly to tumour cells without causing damage to non-tumour cells.

Figure 1. Mechanisms of action of common mAb therapeutics and related products. Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; ADCP, antibody-dependent cellular phagocytosis; CDC, complement-dependent cytotoxicity.