Evaluation of exogenous siRNA addition as a metabolic engineering tool for modifying biopharmaceuticals.

Traditional metabolic engineering approaches, including homologous recombination, zinc-finger nucleases, and short hairpin RNA, have previously been used to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors. This approach has the following potential advantages: no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody (MAb) afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti-CD20 MAb. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17-fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an Antibody-Dependent Cellular Cytoxicity (ADCC) assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods.

T-cadherin structures reveal a novel adhesive binding mechanism.

Vertebrate genomes encode 19 classical cadherins and about 100 nonclassical cadherins. Adhesion by classical cadherins depends on binding interactions in their N-terminal EC1 domains, which swap N-terminal beta-strands between partner molecules from apposing cells. However, strand-swapping sequence signatures are absent from nonclassical cadherins, raising the question of how these proteins function in adhesion. Here, we show that T-cadherin, a glycosylphosphatidylinositol (GPI)-anchored cadherin, forms dimers through an alternative nonswapped interface near the EC1-EC2 calcium-binding sites. Mutations within this interface ablate the adhesive capacity of T-cadherin. These nonadhesive T-cadherin mutants also lose the ability to regulate neurite outgrowth from T-cadherin-expressing neurons. Our findings reveal the likely molecular architecture of the T-cadherin homophilic interface and its requirement for axon outgrowth regulation. The adhesive binding mode used by T-cadherin may also be used by other nonclassical cadherins.

Dynamic properties of a type II cadherin adhesive domain: implications for the mechanism of strand-swapping of classical cadherins.

Cadherin-mediated cell adhesion is achieved through dimerization of cadherin N-terminal extracellular (EC1) domains presented from apposed cells. The dimer state is formed by exchange of N-terminal beta strands and insertion of conserved tryptophan indole side chains from one monomer into hydrophobic acceptor pockets of the partner molecule. The present work characterizes individual monomer and dimer states and the monomer-dimer equilibrium of the mouse Type II cadherin-8 EC1 domain using NMR spectroscopy. Limited picosecond-to-nanosecond timescale dynamics of the tryptophan indole moieties for both monomer and dimer states are consistent with well-ordered packing of the N-terminal beta strands intramolecularly and intermolecularly, respectively. However, pronounced microsecond-to-millisecond timescale dynamics of the side chains are observed for the monomer but not the dimer state, suggesting that monomers transiently sample configurations in which the indole moieties are exposed. The results suggest possible kinetic mechanisms for EC1 dimerization.

Structural analysis of the plakin domain of bullous pemphigoid antigen1 (BPAG1) suggests that plakins are members of the spectrin superfamily.

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins. The plakins are multi-domain proteins that have been shown to interact with microtubules, actin filaments and intermediate filaments, as well as proteins found in cellular junctions. These interactions are mediated through different domains on the plakins. The interactions between plakins and components of specialized cell junctions such as desmosomes and hemidesmosomes are mediated through the so-called plakin domain, which is a common feature of the plakins. We report the crystal structure of a stable fragment from BPAG1, residues 226-448, defined by limited proteolysis of the whole plakin domain. The structure, determined by single-wavelength anomalous diffraction phasing from a selenomethionine-substituted crystal at 3.0 A resolution, reveals a tandem pair of triple helical bundles closely related to spectrin repeats. Based on this structure and analysis of sequence conservation, we propose that the architecture of plakin domains is defined by two pairs of spectrin repeats interrupted by a putative Src-Homology 3 (SH3) domain.

Dye-induced aggregation of single stranded RNA: a mechanistic approach.

The binding of proflavine (D) to single stranded poly(A) (P) was investigated at pH 7.0 and 25 degrees C using T-jump, stopped-flow and spectrophotometric methods. Equilibrium measurements show that an external complex PD(I) and an internal complex PD(II) form upon reaction between P and D and that their concentrations depend on the polymer/dye concentration ratio (C(P)/C(D)). For C(P)/C(D)<2.5, cooperative formation of stacks external to polymer strands prevails (PD(I)). Equilibria and T-jump experiments, performed at I=0.1M and analyzed according to the Schwarz theory for cooperative binding, provide the values of site size (g=1), equilibrium constant for the nucleation step (K( *)=(1.4+/-0.6)x10(3)M(-1)), equilibrium constant for the growth step (K=(1.2+/-0.6)x10(5)M(-1)), cooperativity parameter (q=85) and rate constants for the growth step (k(r)=1.2x10(7)M(-1)s(-1), k(d)=1.1 x 10(2)s(-1)). Stopped-flow experiments, performed at low ionic strength (I=0.01 M), indicate that aggregation of stacked poly(A) strands do occur provided that C(P)/C(D)<2.5.

Type II cadherin ectodomain structures: implications for classical cadherin specificity.

Type I and II classical cadherins help to determine the adhesive specificities of animal cells. Crystal-structure determination of ectodomain regions from three type II cadherins reveals adhesive dimers formed by exchange of N-terminal beta strands between partner extracellular cadherin-1 (EC1) domains. These interfaces have two conserved tryptophan side chains that anchor each swapped strand, compared with one in type I cadherins, and include large hydrophobic regions unique to type II interfaces. The EC1 domains of type I and type II cadherins appear to encode cell adhesive specificity in vitro. Moreover, perturbation of motor neuron segregation with chimeric cadherins depends on EC1 domain identity, suggesting that this region, which includes the structurally defined adhesive interface, encodes type II cadherin functional specificity in vivo.

Structural analysis of mycobacterial and murine hsp60 epitopes in complex with the class I MHC molecule H-2Db.

The decameric peptide SALQNAASIA from the Mycobacterium bovis heat shock protein (hsp) 60 is recognized by the murine T-cell receptor UZ-3-4 in complex with the murine class I major histocompatibility complex molecule H-2D(b). This T-cell receptor cross-reacts with the H-2D(b)-bound non-homologous decameric peptide KDIGNIISDA from the murine hsp60, but does not recognize the nonameric mycobacterial peptide SALQNAASI. Cross-recognition of the KDIGNIISDA/H-2D(b) complex induces autoimmune pathology in immunodeficient mice. We solved the X-ray crystal structure of the SALQNAASIA/H-2D(b) complex at 3.0 A resolution, and we modelled the KDIGNIISDA and SALQNAASI peptides in the H-2D(b) binding site. The structural analysis of the H-2D(b)-bound hsp60 epitopes offers insight into T-cell receptor cross-reactivity.

Amino acid dimorphism and parasite immune evasion: cellular immune responses to a promiscuous epitope of Plasmodium falciparum merozoite surface protein 1 displaying dimorphic amino acid polymorphism are highly constrained.

Like most other surface-exposed antigens of Plasmodium falciparum, the leading malaria vaccine candidate merozoite surface protein (MSP)-1 contains a large number of dimorphic amino acid positions. This type of diversity is presumed to be associated with parasite immune evasion and represents one major obstacle to malaria subunit vaccine development. To understand the precise role of antigen dimorphism in immune evasion, we have analyzed the flexibility of CD4 T cell immune responses against a semi-conserved sequence stretch of the N-terminal block of MSP-1. While this sequence contains overlapping promiscuous T cell epitopes and is a target for growth inhibitory antibodies, three dimorphic amino acid positions may limit its suitability as component of a multi-epitope malaria vaccine. We have analyzed the CD4 T cell responses in a group of human volunteers immunized with a synthetic malaria peptide vaccine containing a single MSP-143-53 sequence variant. All human T cell lines and HLA-DR- or -DP-restricted T cell clones studied were exclusively specific for the sequence variant used for immunization. Competition peptide binding assays with affinity-purified HLA-DR molecules indicated that dimorphism does not primarily affect HLA binding. Modeling studies of the dominant restricting HLA-DRB1*0801 molecule showed that the dimorphic amino acids represent potential TCR contact residues. Lack of productive triggering of the TCR by MHC/variant peptide ligand complexes thus seems to be the characteristic feature of parasite immune evasion associated with antigen dimorphism.