Toward Drug-Like Multispecific Antibodies by Design

Abstract

The success of antibody therapeutics is strongly influenced by their multifunctional nature that couples antigen recognition mediated by their variable regions with effector functions and half-life extension mediated by a subset of their constant regions. Nevertheless, the monospecific IgG format is not optimal for many therapeutic applications, and this has led to the design of a vast number of unique multispecific antibody formats that enable targeting of multiple antigens or multiple epitopes on the same antigen. Despite the diversity of these formats, a common challenge in generating multispecific antibodies is that they display suboptimal physical and chemical properties relative to conventional IgGs and are more difficult to develop into therapeutics. Here we review advances in the design and engineering of multispecific antibodies with drug-like properties, including favorable stability, solubility, viscosity, specificity and pharmacokinetic properties. We also highlight emerging experimental and computational methods for improving the next generation of multispecific antibodies, as well as their constituent antibody fragments, with natural IgG-like properties. Finally, we identify several outstanding challenges that need to be addressed to increase the success of multispecific antibodies in the clinic.

Keywords: affinity; aggregation; bispecific; developability; immunogenicity; non-specific binding; pharmacokinetics; polyspecificity; protein engineering; self-association; solubility; specificity; stability; viscosity.

Figure 1

Antigen-binding regions of monospecific IgGs can be combined into various multispecific formats that have unique developability concerns, including those related to their stability, solubility, aggregation, viscosity, and pharmacokinetics. The abbreviations are scFv-IgG for single-chain variable fragment immunoglobulin, DVD-Ig for dual variable domain immunoglobulin, FIT-Ig for fabs-in-tandem immunoglobulin, BiTE for bispecific T-cell engager and DART for dual-affinity re-targeting antibody fragment.

Figure 2

Evaluation and prediction of mutations in the V_H/V_L interface that enhance the stability and affinity of single-chain antibodies. Deep mutational analysis of a scFv library (top left) identified eight interface residues tolerant of mutations (purple, top right). Combinations of affinity-maintaining mutations at these sites were analyzed with Rosetta to predict those that also improve thermal stability. After development using the anti-lysozyme antibody D44.1, the automated design method (AbLift) was validated on two additional antibodies, including the anti-VEGF antibody G6 (bottom right). AbLift designed G6 variants 1 (V1) and 2 (V2) displayed both improved affinity and thermal stability (bottom left). The figure is adapted from a previous publication [60].

Figure 3

Evaluation of the role of chemical modifications (deamidation) on Fc-mediated receptor binding for bispecific antibodies. Bispecific mAbs were generated with asymmetric levels of deamidation and evaluated for their binding to FcγRIII. (A) mAbs 1 and 2 were heat stressed for 4 months to force deamidation in the Fc region (Asn 330). (B) Bispecific mAbs with various levels of symmetric (BsAb5, BsAb 8) or asymmetric (BsAb6, BsAb7) deamidation were produced through Fab-arm exchange of pooled native and stressed mAbs. The resulting bispecific antibodies display reduced levels of FcγRIII binding relative to BsAb5 in a manner consistent with total levels of deamidation. The figure is adapted from a previous publication [128].

Figure 4

Impact of an additional intramolecular disulfide bond in single-chain antibody fragments, either in isolation or as IgG-scFv bispecific antibodies, on their biophysical properties. Comparison of an anti-IL17 scFv with and without an additional intramolecular (H44-L100) disulfide bond as isolated scFvs or IgG-scFv fusions. (A) Differential scanning calorimetry (DSC) thermogram and (B) analytical SEC chromatograms for the wild-type scFv. (C) DSC thermogram and (D) analytical SEC chromatograms for the disulfide-stabilized scFv. (E) DSC thermogram of the disulfide-stabilized bispecific antibody. (F) ANS fluorescence emission (300 µM ANS) of the wild-type (blue) and disulfide-stabilized (purple) scFvs. In (A,C,E), the raw data are shown in grey, and the fitted data are shown in color (blue, purple or green). The figure is adapted from a previous publication [155].

Figure 5

Evaluation of the viscoelastic properties of bispecific antibodies and approaches for reducing their viscosities using formulation and mutational strategies. (A) Structures of anti-IL13/IL17 mono- and bi-specific antibodies, the latter of which were generated using knob-in-hole heavy chain pairing methods. (B–D) Measured viscosities at 150 mg/mL in standard formulation conditions (20 mM His-HCl, pH 6.0) for (B) various antibody formats, (C) IgGs at different formulation conditions, and (D) IgGs with various point mutations in the anti-IL13 variable domains. The viscosity of one of the parental IgGs is shown with a grey dotted line. The figure is adapted from a previous publication [174].

Figure 6

Effect of molecular architecture of IgG fusion proteins on their hydrophobicities, stabilities, and pharmacokinetic properties. (A) Representations of various IgG-extracellular domains (ECDs) with the D2 domain of VEGFR1 fused to different regions of the wild-type IgG. (B) Hydrophobicity measured by hydrophobic interaction chromatography (HIC; blue) and stability (first thermal unfolding transition, red) of the wild-type (WT) IgG and IgG-ECDs. (C) Pharmacokinetic profiles of the WT IgG and IgG-ECDs in cynomolgus monkeys (single 2 mg/kg iv dose) reveal increased clearance rates for more hydrophobic formats. The figure is adapted from a previous publication [219].