A single homogeneous assay for simultaneous measurement of bispecific antibody target binding.

Bispecific antibodies (BsAbs) are engineered to simultaneously bind two different antigens, and offer promising clinical outcomes for various diseases. The dual binding properties of BsAbs may enable superior efficacies and/or potencies compared to standard monoclonal antibodies (mAbs) or combination mAb therapies. Characterizing BsAb binding properties is critical during biotherapeutic development, where data is leveraged to predict efficacy and potency, assess critical quality attributes and improve antibody design. Traditional single-target, single-readout approaches (e.g., ELISA) have limited usefulness for interpreting complex bispecific binding, and double the benchwork. To address these deficiencies, we developed and implemented a new dual-target/readout binding assay that accurately dissects the affinities of both BsAb binding domains directly and simultaneously. This new assay uses AlphaPlex® technology, which eliminates traditional ELISA wash steps and can be miniaturized for automated workflows. The optimized BsAb AlphaPlex assay demonstrates 99-107% accuracy within a 50-150% linear range, and detected >50% binding degradation from photo- and thermal stress conditions. To the best of our knowledge, this is the first instance of a dual-target/readout BsAb AlphaPlex assay with GMP-suitable linear range, accuracy, specificity, and stability-indicating properties. As a highly customizable and efficient assay, BsAb AlphaPlex may be applicable to numerous bispecific formats and/or co-formulations against a variety of antigens beyond the clinical therapeutic space.

Prevention of Fab-arm exchange and antibody reduction via stabilization of the IgG4 hinge region.

IgG4s are dynamic molecules that undergo a process called Fab-arm exchange. Disulfide bonds between heavy chains are transiently reduced, resulting in half antibodies that reform intact antibodies with other IgG4 half antibodies. In vivo, therapeutic IgG4s can recombine with endogenous IgG4s, resulting in a heterogeneous mixture of bispecific antibodies. A related issue that can occur for any therapeutic protein during manufacturing is interchain disulfide bond reduction. For IgG4s, this primarily results in high levels of half-mAb that persist through purification processes. The S228P mutation has been used to prevent half-mAb formation. However, we demonstrated that IgG4s with the S228P mutation are subject to half-mAb formation and Fab-arm exchange in reducing environments. We identified two novel mutations that stabilize the heavy-heavy chain interaction via incorporation of additional disulfide bonds in the hinge region. Individually, these mutations increase stability toward reduction and lessen Fab-arm exchange. Combination of all three mutations, Y219C, G220C, and S228P, has an additive benefit resulting in an IgG4 with ˃7-fold increase in stability toward reduction while preventing Fab-arm exchange. Importantly, the mutations do not affect antigen binding or Fc effector function. These mutations hold great promise for solving mAb reduction during manufacturing and preventing Fab-arm exchange in vivo.

Potent dengue virus neutralization by a therapeutic antibody with low monovalent affinity requires bivalent engagement.

We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 Šresolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface.

Atomic-level functional model of dengue virus Envelope protein infectivity.

A number of structures have been solved for the Envelope (E) protein from dengue virus and closely related flaviviruses, providing detailed pictures of the conformational states of the protein at different stages of infectivity. However, the key functional residues responsible for mediating the dynamic changes between these structures remain largely unknown. Using a comprehensive library of functional point mutations covering all 390 residues of the dengue virus E protein ectodomain, we identified residues that are critical for virus infectivity, but that do not affect E protein expression, folding, virion assembly, or budding. The locations and atomic interactions of these critical residues within different structures representing distinct fusogenic conformations help to explain how E protein (i) regulates fusion-loop exposure by shielding, tethering, and triggering its release; (ii) enables hinge movements between E domain interfaces during triggered structural transformations; and (iii) drives membrane fusion through late-stage zipper contacts with stem. These results provide structural targets for drug and vaccine development and integrate the findings from structural studies and isolated mutagenesis efforts into a cohesive model that explains how specific residues in this class II viral fusion protein enable virus infectivity.