Engineering therapeutic monoclonal antibodies

Abstract

The use of human antibodies as biologic therapeutics has revolutionized patient care throughout fields of medicine. As our understanding of the many roles antibodies play within our natural immune responses continues to advance, so will the number of therapeutic indications for which an mAb will be developed. The great breadth of function, long half-life, and modular structure allow for nearly limitless therapeutic possibilities. Human antibodies can be rationally engineered to enhance their desired immune functions and eliminate those that may result in unwanted effects. Antibody therapeutics now often start with fully human variable regions, either acquired from genetically engineered humanized mice or from the actual human B cells. These variable genes can be further engineered by widely used methods for optimization of their specificity through affinity maturation, random mutagenesis, targeted mutagenesis, and use of in silico approaches. Antibody isotype selection and deliberate mutations are also used to improve efficacy and tolerability by purposeful fine-tuning of their immune effector functions. Finally, improvements directed at binding to the neonatal Fc receptor can endow therapeutic antibodies with unbelievable extensions in their circulating half-life. The future of engineered antibody therapeutics is bright, with the global mAb market projected to exhibit compound annual growth, forecasted to reach a revenue of nearly half a trillion dollars in 2030.

Keywords: Allergy; IgE; antibody engineering; immunology; mAb; therapeutic antibody.

Figure 1.. Antibody structure, isotype/subtypes, and their major features.

The structure of a human antibody can be broken into two basic parts linked by a hinge, helping separate its functional regions. The antigen-binding fragment (Fab) contains the antibody variable region of the antibody, which is the product of gene rearrangement and somatic hypermutation, allowing for highly specific antigen binding. The constant or crystallizable fragment (Fc), comes in a variety of types, known as isotypes (there are five isotypes: IgM, IgD, IgG, IgA and IgE), and is responsible for binding complement and a diversity of cell receptors (FcRs), resulting in many immune functions. Immune functions are influenced by the structural differences existing between the isotypes and subtypes of antibodies. These differences influence both antigen binding, such as hinge flexibility, and receptor selection, such as glycosylation. The vast array of immune functions, conveyed in an antigen specific fashion, is controlled by these many features of human antibodies.

Figure 2.. Fc receptor profiles for effector cell induction and inhibition.

Receptors bound by IgG antibodies differ from one another in their function, cellular distribution, glycosylation, and affinity to which IgG Fc subtypes they bind. Originally named for its role in IgG transport from the mother to the fetus or neonate, FcRn binds to, transports, and recycles IgG, playing a central role in cellular trafficking and serum half-life of IgG antibodies. FcRn is expressed primarily on vascular endothelial cells but also on epithelial cells, macrophages, and other cell types. Fc receptors present on immune effector cells varies greatly by cell type, but are roughly categorized into three groups: FcγRI, FcγRII, and FcγRIII. FcγRI, FcγRIII and FcγRIIa receptors are capable of activating functions within the cell though ITAM cytoplasmic domains – highlighted in red. Only the FcγRIIb receptor can modulate effects of the other Fc receptors by transmitting an inhibitory signal through their ITIM cytoplasmic domain – highlighted in green. Most of the cellular functions arise through low affinity interactions between Fc and FcRs, requiring multivalent immune complexes for signal transduction to occur, allowing for an additional layer of control. The summation of signal generated by the diversity of interactions ultimately dictate the activation state of the effector cell.

Figure 3.. Fc engineering features for enhancement of therapeutic effects.

Engineering the Fc region of a therapeutic monoclonal antibody allows for generation of unique drugs rationally designed for a strategic pharmacological purpose. Alterations of Fc affinity, such as the engineered YTE mutation, for FcRn can have dramatic effects on serum half-life by greatly facilitating antibody recycling. Simple changes can be performed which result in greater stability and prevention of arm-swapping often seen as a problem for IgG4 subtype antibodies frequently used for their natural tendency to be minimally inflammatory. Modification of Fc residues can result in dramatic increases or decreases in various effector cell functions and their tendency to fix complement. Glycan alteration can also be engineered to improve pharmacokinetics, efficacy, and safety of therapeutic antibodies. A wide array of Fc engineering features must be considered carefully when designing antibodies for therapeutic use.

Figure 4.. Rational engineering of theoretical allergen blocking therapeutics.

The IgE molecule bound to its high affinity receptor provides specific control for the release of the mediators responsible for the symptoms of allergic diseases. Blocking antibody therapeutics, created from the variable sequences of allergen specific IgE, in theory can be engineered for the purpose of passive immunotherapy, to mitigate against anaphylaxis, or augmenting immunotherapy, to foster immune reprogramming – targeting immune mechanisms that could in theory provide advantages for treating allergy. Such antibodies could have engineered features geared toward enhancement of such overall therapeutic goals: 1. improved affinity for FcRn to increase serum half-life, 2. variable regions taken from an allergen specific IgE binds an IgE epitope to block IgE binding, 3. improved affinity for FcγRIIb to send inhibitory signaling via allergen specific cross-linking or co-aggregation of FcεRI and FcγRIIb, 4. improve antigen uptake/presentation by APCs through FcγRIII, to facilitate reprogramming of the allergen specific adaptive immune response. Use of IgG4 subtype and additional alterations of the Fc could also improve tolerability and reduce potential side-effects caused by complement and unwanted pro-inflammatory effector functions. Additional engineered mAbs, antibody cocktails, or bispecific antibodies may be needed to block sufficient allergen epitopes to prevent allergic symptoms.