Quantifying antibody binding: techniques and therapeutic implications

Abstract

The binding kinetics of an antibody for its target antigen represent key determinants of its biological function and success as a novel biotherapeutic. Defining these interactions and kinetics is critical for understanding the pharmacological and pharmacodynamic profiles of antibodies in therapeutic applications, with line of sight to clinical translation. In this review, we discuss the latest developments in approaches to measure and modulate antibody-antigen interactions, including antibody engineering, novel antibody formats, current, and emerging technologies for measuring antibody-antigen binding interactions, and emerging perspectives within the field. We also explore how emerging computational methods are set to become powerful tools for modeling antibody-binding interactions under physiologically relevant conditions. Finally, we consider the therapeutic implications of modulating target engagement in terms of pharmacodynamics and pharmacokinetics.

Keywords: Antibody; affinity; avidity; pharmacokinetics; pharmacology; target engagement.

Figure 1.

(a) Schematic depiction of the structure of IgG, the most frequently observed therapeutic antibody isotype. The antibody contains two heavy and two light chains, with disulphide bridges connecting the chains. The lower region, consisting of the CH2 and CH3 domains is frequently referred to as the Fc region. The upper region, containing the CH1 and VH regions of the heavy chain, and CL and VL regions of the light chain, are referred to as the Fab region. The VH and VL regions specifically include CDRs, which are key modulators of antibody target specificity and binding. These are separated by a flexible hinge region. (b) A selection of the best-represented antibody-derived therapeutic formats under investigation.

Figure 2.

Antibodies are bivalent and can bind multiple targets through avidity. For monospecific antibodies, both arms have the same affinity, and the strength of the second binding event is enhanced by the constraint imposed by the primary binding event.

Figure 3.

Schematic overview of phage display. (a). Libraries of bacteriophage expressing surface antibodies are generated from the transfection of bacteria with phagemids containing antibody coding sequences. (b). A target of interest is immobilised, and the naïve phage library applied. (c). Phages encoding non-specific antibodies are washed off while those encoding target specific antibodies remain bound. (d). Bound phages are eluted, amplified, and analysed. Relative binding affinity may be analysed using flow cytometry and antibody encoding nucleic acids sequenced. (e). 3-5 rounds of biopanning are commonly performed in which high affinity antibodies re-enter the phage display cycle until sufficiently enriched.⁴⁶

Figure 4.

(a) Schematic of a cis binding mode, in which both Fab domains engage with two cell surface receptors, or two domains on a single protein. (b) Schematic of a trans binding mode, in which both Fab domains engage with antigens on distinct cells, or two distinct antigens in solution. (c) Schematic of a trispecific antibody, engaging in cis on the target cell, and engaging in trans on the effector cell using a covalently bound Fab. (d) A biparatopic antibody engaging two epitopes on the same target, both within the same antigen, and bridging between two antigens. (e) An immunocytokine binding in cis on the target cell, used to selectively target cytokine engagement also in cis.

Figure 5.

Schematic of an SPR chip surface in two assay design formats. In assay A, the binding partner is covalently immobilised onto the chip surface, with the antibody introduced in solution. Assay B shows the experiment in an alternative format in which the antibody is immobilised onto the chip surface, with the binding partner in solution.

Figure 6.

A summary of antibody mechanisms of action. Antibodies can elicit a range of effects, both directly for example by direct blockade of ligand binding to receptor or by inducing receptor clustering with subsequent activation of downstream signalling events, or indirectly by recruiting effector cells to the target cell as illustrated here by the recruitment of NK cells or phagocytes such as macrophages or neutrophils to induce target cell killing by ADCC or ADCP, respectively. Further cytotoxic mechanisms can be triggered by recruitment of complement to the cell surface or by engagement with alternative effector cells such as T cells. Antibodies can also be used to deliver a ‘payload’, commonly a toxin to target and kill tumour cells, by forming an ADC.

Figure 7.

Schematic of an antibody-drug-conjugate and their requisite components.