Antibody Aggregation: Insights from Sequence and Structure

Abstract

Abstract:Monoclonal antibodies (mAbs) are the fastest-growing biological therapeutics with important applications ranging from cancers, autoimmunity diseases and metabolic disorders to emerging infectious diseases. Aggregation of mAbs continues to be a major problem in their developability. Antibody aggregation could be triggered by partial unfolding of its domains, leading to monomer-monomer association followed by nucleation and growth. Although the aggregation propensities of antibodies and antibody-based proteins can be affected by the external experimental conditions, they are strongly dependent on the intrinsic antibody properties as determined by their sequences and structures. In this review, we describe how the unfolding and aggregation susceptibilities of IgG could be related to their cognate sequences and structures. The impact of antibody domain structures on thermostability and aggregation propensities, and effective strategies to reduce aggregation are discussed. Finally, the aggregation of antibody-drug conjugates (ADCs) as related to their sequence/structure, linker payload, conjugation chemistry and drug-antibody ratio (DAR) is reviewed.

Considerations for the Design of Antibody-Based Therapeutics

“…Aggregation is closely coupled to stability. 173,174,299,300 While all proteins undergo continuous conformational sampling, less stable proteins are more likely to partially unfold and reveal hydrophobic residues that are buried in the native state. Transient exposure of hydrophobic, uncharged patches allows for intermolecular association of these regions.…”

“…Because aggregation of this sort locks proteins in nonnative conformations, it is often considered to be irreversible. 300 Some regions of antibodies are more likely than others to initiate aggregation. The intradomain and interdomain contacts, such as those between V H and V L domains, are especially prone to aggregation because of their hydrophobic character.…”

“…For this reason, sdAbs (V H , V L ) are often engineered to reduce hydrophobicity at the normal domain interface, and scFvs may be modified to minimize transient opening that can lead to aggregation. [300][301][302] IgG binding sites (CDRs for antigen binding, lower hinge and upper Cg2 for FcgR and C1q binding, and the Cg2/Cg3 elbow for FcRn binding) also tend to have hydrophobic residues that contribute to the energy of binding. 300 For ADCs and other conjugates, hydrophobic linkers or payloads have the potential to increase aggregation.…”

“…[300][301][302] IgG binding sites (CDRs for antigen binding, lower hinge and upper Cg2 for FcgR and C1q binding, and the Cg2/Cg3 elbow for FcRn binding) also tend to have hydrophobic residues that contribute to the energy of binding. 300 For ADCs and other conjugates, hydrophobic linkers or payloads have the potential to increase aggregation. 300 Although IgG molecules are considered especially stable proteins, efforts to improve developability have focused on protein engineering and formulation strategies to further reduce the incidence of aggregation.…”

High Affinity Maturated Human Antibodies from Naïve and Synthetic Antibody Repertoires

“…The antigen-binding site of an antibody consists of the surrounding framework regions and the complementarity determining regions (CDRs), CDR1, CDR2 and CDR3. CDR3 region are particularly important for antibody-antigen specificity [43]. The V(D)J and V(J) rearrangement of the antibody gene segments and somatic mutations will give rise to higher binding diversities to various antigens [35,36].…”