In vivo pharmacokinetic enhancement of monomeric Fc and monovalent bispecific designs through structural guidance.

In a biologic therapeutic landscape that requires versatility in targeting specificity, valency and half-life modulation, the monomeric Fc fusion platform holds exciting potential for the creation of a class of monovalent protein therapeutics that includes fusion proteins and bispecific targeting molecules. Here we report a structure-guided approach to engineer monomeric Fc molecules to adapt multiple versions of half-life extension modifications. Co-crystal structures of these monomeric Fc variants with Fc neonatal receptor (FcRn) shed light into the binding interactions that could serve as a guide for engineering the half-life of antibody Fc fragments. These engineered monomeric Fc molecules also enabled the generation of a novel monovalent bispecific molecular design, which translated the FcRn binding enhancement to improvement of in vivo serum half-life.

Experimentally guided computational antibody affinity maturation with de novo docking, modelling and rational design.

Antibodies are an important class of therapeutics that have significant clinical impact for the treatment of severe diseases. Computational tools to support antibody drug discovery have been developing at an increasing rate over the last decade and typically rely upon a predetermined co-crystal structure of the antibody bound to the antigen for structural predictions. Here, we show an example of successful in silico affinity maturation of a hybridoma derived antibody, AB1, using just a homology model of the antibody fragment variable region and a protein-protein docking model of the AB1 antibody bound to the antigen, murine CCL20 (muCCL20). In silico affinity maturation, together with alanine scanning, has allowed us to fine-tune the protein-protein docking model to subsequently enable the identification of two single-point mutations that increase the affinity of AB1 for muCCL20. To our knowledge, this is one of the first examples of the use of homology modelling and protein docking for affinity maturation and represents an approach that can be widely deployed.

Developability Assessment of Engineered Monoclonal Antibody Variants with a Complex Self-Association Behavior Using Complementary Analytical and in Silico Tools.

Monoclonal antibodies (mAbs) are complex molecular structures. They are often prone to development challenges particularly at high concentrations due to undesired solution properties such as reversible self-association, high viscosity, and liquid-liquid phase separation. In addition to formulation optimization, applying protein engineering can provide an alternative mitigation strategy. Protein engineering during the discovery phase can provide great benefits to optimize molecular properties, resulting in improved developability profiles. Here, we present a case study utilizing complementary analytical and predictive in silico methods. We have systematically identified and reengineered problematic residues responsible for the self-association of a model mAb, driven by a complex combination of hydrophobic and electrostatic interactions. Noteworthy findings include a more dominant contribution of hydrophobic interactions to self-association and potential feasibility of mutations in the CDR regions to mitigate self-association. The engineered mutation panel enabled us to assess potential correlations among commonly utilized developability screening assays, including affinity capture self-interaction nanospectroscopy (AC-SINS), dynamic light scattering (DLS), and apparent solubility by PEG-precipitation. In addition, we evaluated the correlations between experimental measurements and computational predictions. CamSol, an in silico computational tool that accounts for complex molecular interactions and neighboring hotspots, was found to be an effective screening tool. Our work led to reengineered mAb variants, better suited for high-concentration liquid formulation development. The engineered mAbs exhibited enhanced in vitro and simulated in vivo solubility and reduced self-association propensity, while maintaining binding affinity and thermal stability.

Generation and Characterization of an IgG4 Monomeric Fc Platform.

The immunoglobulin Fc region is a homodimer consisted of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and extended serum half-life. However, attempts to generate one-arm fusion proteins with monomeric Fc, with one set of CH2 and CH3 domains, are often plagued with challenges such as weakened binding to FcRn or partial monomer formation. Here, we demonstrate the generation of a stable IgG4 Fc monomer with a unique combination of mutations at the CH3-CH3 interface using rational design combined with in vitro evolution methodologies. In addition to size-exclusion chromatography and analytical ultracentrifugation, we used multi-angle light scattering (MALS) to show that the engineered Fc monomer exhibits excellent monodispersity. Furthermore, crystal structure analysis (PDB ID: 5HVW) reveals monomeric properties supported by disrupted interactions at the CH3-CH3 interface. Monomeric Fc fusions with Fab or scFv achieved FcRn binding and serum half-life comparable to wildtype IgG. These results demonstrate that this monomeric IgG4 Fc is a promising therapeutic platform to extend the serum half-life of proteins in a monovalent format.