Considerations for the Design of Antibody-Based Therapeutics

Abstract

Antibody-based proteins have become an important class of biologic therapeutics, due in large part to the stability, specificity, and adaptability of the antibody framework. Indeed, antibodies not only have the inherent ability to bind both antigens and endogenous immune receptors but also have proven extremely amenable to protein engineering. Thus, several derivatives of the monoclonal antibody format, including bispecific antibodies, antibody-drug conjugates, and antibody fragments, have demonstrated efficacy for treating human disease, particularly in the fields of immunology and oncology. Reviewed here are considerations for the design of antibody-based therapeutics, including immunological context, therapeutic mechanisms, and engineering strategies. First, characteristics of antibodies are introduced, with emphasis on structural domains, functionally important receptors, isotypic and allotypic differences, and modifications such as glycosylation. Then, aspects of therapeutic antibody design are discussed, including identification of antigen-specific variable regions, choice of expression system, use of multispecific formats, and design of antibody derivatives based on fragmentation, oligomerization, or conjugation to other functional moieties. Finally, strategies to enhance antibody function through protein engineering are reviewed while highlighting the impact of fundamental biophysical properties on protein developability.

Keywords: IgG antibody(s); antibody drug conjugate(s) (ADC); antibody drug(s); antibody(s); developability; drug design; immunotherapy; monoclonal antibody(s); pharmacokinetics; physicochemical properties.

Figure 1:

Structural considerations for the design of IgG-based therapeutics and their effects on biological and clinical function.

Figure 2:

Strategies for identification of antibody variable regions. Rearranged V(D)J genes may be sourced from the spleens of immunized animals (1a), or from the blood of naïve, vaccinated, or chronically ill patients (1b). Synthetic DNA libraries have also been created with an emphasis on diversity within complementarity determining regions. After isolating B cells, the variable regions of their B cell receptors are used to generate surface-expressed antibody domains that may be used in functional screens. One approach is to immortalize the B cells via fusion with myeloma cells, producing highly proliferative antibody-producing hybridomas (2a). Alternatively, mRNA can be isolated from B cells, converted to cDNA, and used to construct libraries of phage, bacteria, yeast, or mammalian cells with surface display of Fab or scFv fragments (2b). The repertoire of B cells may also be directly sequenced via Ig seq or sorted/diluted into single B cell populations for sequencing (2c). Whether using hybridoma, surface display, or (single) B cells, screening steps are used to select for functional antigen binders. This selection may occur at the cell level using FACS (3a) or at the protein level using ELISA of cell supernatants (3b). After enrichment of functional cells or proteins, successful candidates may be identified at the DNA (4a) or protein (4b) levels. While cloning, selection, and sequencing are required steps for identification of functional Ig genes, they may be performed in orders other than those represented here.

Figure 3:

Common post-translational modifications of IgG antibodies. Shown on the left are amino acid modifications that occur in a side-chain dependent but site-independent manner. These chemical alterations may negatively affect properties like antigen or receptor binding. Shown on the right are amino acid modifications that occur at specific sites. While N-terminal formation of pyroglutamate occurs only for chains that begin with glutamine or glutamate, C-terminal lysine clipping occurs for all IgG antibodies, whose heavy chains terminate with a glycine-lysine motif. Glycosylation at asparagine 297 leads to a core glycan (solid lines) to which additional sugars may be added (dotted lines). These differences in glycan composition have significant effects on binding to Fc receptors. Abbreviations: Bis-GlcNAc (bisecting N-acetylglucosamine), Fuc (fucose), Gal (galactose), Man (mannose), Neu5Ac (N-acetylneuraminic acid), SA (sialic acid)

Figure 4:

Therapeutic frameworks based on fragmentation, multimerization, conjugation, and fusion of human or non-human antibody domains. Abbreviations: ADC (antibody-drug conjugate), ARC (antibody-radionuclide conjugate), Fab (antigen-binding fragment), Fc (crystallizable fragment), hcAb (heavy chain antibody), IgG (immunoglobulin G), PEG (polyethylene glycol), scFv (single-chain variable fragment), sdAb (single domain antibody)

Figure 5:

Types of bispecific antibody frameworks. Fragment fusions are small proteins created by genetic fusion of antibody domains (Fab, scFv, sdAb). Their lack of Fc domain confers high diffusion and tissue penetration, but fast clearance and lack of effector function. Asymmetric IgG frameworks retain many properties of native IgG but bind distinct antigens via each Fab arm. Fusion of antibody fragments to the IgG framework creates large, generally symmetric molecules that are often multivalent.

Figure 6:

Thermodynamics of antibody folding and binding in dilute buffer and in complex matrices like the serum. Because the free energy of unfolded, native, and bound antibodies may differ significantly in buffer and in serum, the stability (ΔG_folding) and binding affinity (ΔG_binding) may also differ in these two types of media. Thus, it is important to characterize and understand the thermodynamic properties of antibodies in complex but biologically relevant environments.