Impact of antibody Fc engineering on translational pharmacology, and safety: insights from industry case studies

Abstract

Therapeutic monoclonal antibodies (mAbs) are often designed to not only bind targets via their antigen-binding domains (Fabs) but to also engage with cell surface receptors, FcγRs and FcRn, through their Fc regions, which may result in a variety of functional outcomes, including antibody- dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and alteration of circulating half-lives. Engineering the Fc regions to achieve desirable pharmacology and pharmacokinetics is a widely adopted strategy in drug development. Fc regions can be modified through amino acid substitutions and glycoengineering, resulting in enhanced or reduced effector functions, preferential binding to FcR subtypes, or pH-dependent binding to FcRns. These alterations in binding and effector activities of mAbs may potentially also be accompanied by undesirable effects or safety concerns. Critical assessment of pharmacology and safety in the nonclinical setting is essential before exposing humans to the engineered mAb. For Fc-modified mAbs, the choice of in vitro and in vivo nonclinical pharmacology and safety models need to account for species differences in FcR expression and function, potentially divergent effects of Fc modifications in humans versus nonclinical species, impact of target and cognate ligand expression patterns, and potential impact of emergent anti-drug antibodies directed against the mAb. Using a variety of industry case studies, we highlight key aspects of nonclinical pharmacology and toxicology testing strategies, factors that influence choice of nonclinical models, translatability of findings, input from health authorities and suggest best practice approaches for nonclinical testing of Fc modified mAbs.

Keywords: Effector function; Fc enhancement; Fc receptor blockade; Fc receptors; Fc silencing; monoclonal antibody.

Figure 1.

Key Fc-mediated effector functions of IgG. (a) Fc binding to activating FcγRs on NK cells and macrophages to promote their activation and mediation of antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP), respectively. Fc binding to C1q to mediate complement activation and direct complement-mediated cell cytotoxicity (CDC), and via C3b generation, opsonizing of target cells for complement-mediated cell cytotoxicity (CDCC) via the C3b receptor (C3bR) on macrophages and neutrophils. (b) Binding of the Fc within IgG-antigen immune complexes to the inhibitory FcγRIIB on B cells, NK cells and macrophages to inhibit their activation via the B cell receptor (BCR) or activating FcyRs, respectively. (c) Binding of the Fc to FcγRIIB on B cells which acts as a scaffold to enhance cross-linking of Fab-bound receptors on T cells and other cells leading to target-cell activation or apoptosis depending on the receptor type, (d) Binding of the Fc within IgG-antigen immune complexes to FcγRIIB on antigen-presenting cells (APCs) to promote the internalization, clearance, and presentation to T cells of Fab-bound antigen.

Figure 2.

Recycling of IgG by FcRn general schematic of FcRn-mediated recycling of IgG antibodies. Circulating plasma proteins (e.g., antibodies, albumin, cytokines) can undergo fluid phase endocytosis into endosomes. Upon entry into, maturation, and acidification of endosomes, IgG antibodies (red) may bind to FcRn in a pH-dependent manner. Once formed, the FcRn-IgG complex may be recycled to the plasma membrane, where exposure to the neutral pH in circulation facilitates dissociation. Antibodies and serum proteins that are unbound to FcRn or simply cannot bind to FcRn are targeted for lysosomal degradation, where catabolizing enzymes will break down proteins into peptides and amino acids.

Figure 3.

Safety-relevant aspects of enhanced Fc-effector function created in https://biorender.com (A) IgG structure indicating locations of critical immune receptor / ligand interactions. (B) Complement related killing and immune cell activation. The classical complement pathway kills cells directly after engagement on clustered IgG (CDC), effected by membrane attack complex (MAC) or by opsonisation of target cells with complement ‘b’ fragments (or opsonins), which are recognised by phagocytic effector cells (ADCP). Complement ‘a’ fragments (or anaphylotoxins) are highly potent effectors of immune cell infiltration and activation of immune and endothelial cells with potential for further cytokine release. (C) IgG binding to FcRn in endothelial, macrophages and other cells prolongs IgG half-life and hence exposure to its pharmacological activity potentially increasing the risk of pharmacology-associated toxicity. (D). Receptor clustering driven directly by Ab format engineering or facilitated in trans by CD32b on a neighbouring cell can lead to receptor signalling and hence cell activation or inhibition, depending on receptor biology and cellular context. (E) Cellular killing mechanisms effected through FcγRIIIa on NK cells (ADCC) or primarily FcγRIIa on phagocytic cells (ADCP). NK cells secrete local effectors such as perforin, IFNγ, granzyme B to kill cells directly. Phagocytic cells engage and kill target cells, via IgG and / or opsonins to directly ‘eat’ target cells or strip off sections of membrane ‘trogocytosis’. (F) IgG aggregates or target antigen related immune-complexes can activate FcγR resulting in ‘off-target’ / ‘off biology’ related immune mediator / cytokine release toxicology.