Production of stable bispecific IgG1 by controlled Fab-arm exchange

The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production. The bispecific antibody was generated by mixing the two parental molecules under controlled reducing conditions, resulting in efficient Fab-arm exchange of >95% at kg scale. The reductant was removed via diafiltration, resulting in spontaneous reoxidation of interchain disulfide bonds. Aside from the bispecific nature of the molecule, extensive characterization demonstrated that the IgG1 structural integrity was maintained, including function and stability. These results demonstrate the suitability of this bispecific IgG1 format for commercial-scale manufacturing using standard antibody manufacturing techniques.     

Integrating qPLM and biomechanical test data with an anisotropic fiber distribution model and predictions of TGF-\upbeta 1 and IGF-1 regulation of articular cartilage fiber modulus

A continuum mixture model with distinct collagen (COL) and glycosaminoglycan elastic constituents was developed for the solid matrix of immature bovine articular cartilage. A continuous COL fiber volume fraction distribution function and a true COL fiber elastic modulus ( $E^\mathrm{f})$ were used. Quantitative polarized light microscopy (qPLM) methods were developed to account for the relatively high cell density of immature articular cartilage and used with a novel algorithm that constructs a 3D distribution function from 2D qPLM data. For specimens untreated and cultured in vitro, most model parameters were specified from qPLM analysis and biochemical assay results; consequently, $E^\mathrm{f}$ was predicted using an optimization to measured mechanical properties in uniaxial tension and unconfined compression. Analysis of qPLM data revealed a highly anisotropic fiber distribution, with principal fiber orientation parallel to the surface layer. For untreated samples, predicted $E^\mathrm{f}$ values were 175 and 422 MPa for superficial (S) and middle (M) zone layers, respectively. TGF- $\upbeta $ 1 treatment was predicted to increase and decrease $E^\mathrm{f}$ values for the S and M layers to 281 and 309 MPa, respectively. IGF-1 treatment was predicted to decrease $E^\mathrm{f}$ values for the S and M layers to 22 and 26 MPa, respectively. A novel finding was that distinct native depth-dependent fiber modulus properties were modulated to nearly homogeneous values by TGF- $\upbeta $ 1 and IGF-1 treatments, with modulated values strongly dependent on treatment.     

Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons

Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low‐affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin‐positive (PV) interneurons. Nrg3 and ErbB4 are located pre‐ and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo. Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short‐term plasticity, and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4⁺ interneurons and affects short‐term plasticity. Nrg3 mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non‐neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4⁺ interneurons.     

Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial genome

Two novel mitochondrial gene arrangements are identified in an agamid lizard and a ranid frog. Statistical tests incorporating phylogeny indicate a link between novel vertebrate mitochondrial gene orders and movement of the origin of light-strand replication. A mechanism involving errors in light-strand replication and tandem duplication of genes is proposed for rearrangement of vertebrate mitochondrial genes. A second mechanism involving small direct repeats also is identified. These mechanisms implicate gene order as a reliable phylogenetic character. Shifts in gene order define major lineages without evidence of parallelism or reversal. The loss of the origin of light-strand replication from its typical vertebrate position evolves in parallel and, therefore, is a less reliable phylogenetic character. Gene junctions also evolve in parallel. Sequencing across multigenic regions, in particular transfer RNA genes, should be a major focus of future systematic studies to locate novel gene orders and to provide a better understanding of the evolution of the vertebrate mitochondrial genome.      

A bimodular polyconvex anisotropic strain energy function for articular cartilage

A strain energy function for finite deformations is developed that has the capability to describe the nonlinear, anisotropic, and asymmetric mechanical response that is typical of articular cartilage. In particular, the bimodular feature is employed by including strain energy terms that are only mechanically active when the corresponding fiber directions are in tension. Furthermore, the strain energy function is a polyconvex function of the deformation gradient tensor so that it meets material stability criteria. A novel feature of the model is the use of bimodular and polyconvex "strong interaction terms" for the strain invariants of orthotropic materials. Several regression analyses are performed using a hypothetical experimental dataset that captures the anisotropic and asymmetric behavior of articular cartilage. The results suggest that the main advantage of a model employing the strong interaction terms is to provide the capability for modeling anisotropic and asymmetric Poisson's ratios, as well as axial stress-axial strain responses, in tension and compression for finite deformations.