Affinity Improvement of a Therapeutic Antibody by Structure-Based Computational Design: Generation of Electrostatic Interactions in the Transition State Stabilizes the Antibody-Antigen Complex

The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.     

Modeling light adaptation in circadian clock: prediction of the response that stabilizes entrainment

Periods of biological clocks are close to but often different from the rotation period of the earth. Thus, the clocks of organisms must be adjusted to synchronize with day-night cycles. The primary signal that adjusts the clocks is light. In Neurospora, light transiently up-regulates the expression of specific clock genes. This molecular response to light is called light adaptation. Does light adaptation occur in other organisms? Using published experimental data, we first estimated the time course of the up-regulation rate of gene expression by light. Intriguingly, the estimated up-regulation rate was transient during light period in mice as well as Neurospora. Next, we constructed a computational model to consider how light adaptation had an effect on the entrainment of circadian oscillation to 24-h light-dark cycles. We found that cellular oscillations are more likely to be destabilized without light adaption especially when light intensity is very high. From the present results, we predict that the instability of circadian oscillations under 24-h light-dark cycles can be experimentally observed if light adaptation is altered. We conclude that the functional consequence of light adaptation is to increase the adjustability to 24-h light-dark cycles and then adapt to fluctuating environments in nature.     

Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner

Halophilic β-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649–658, 2010b). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus. The “Brevibacillus in vivo cloning” method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution.     

Identification of small-molecule inhibitors of the human S100B–p53 interaction and evaluation of their activity in human melanoma cells

The interaction between human S100 calcium-binding protein B (S100B) and the tumor suppressor protein p53 is considered to be a possible therapeutic target for malignant melanoma. To identify potent inhibitors of this interaction, we screened a fragment library of compounds by means of a fluorescence-based competition assay involving the S100B-binding C-terminal peptide of p53. Using active compounds from the fragment library as query compounds, we constructed a focused library by means of two-dimensional similarity searching of a large database. This simple, unbiased method allowed us to identify several inhibitors of the S100B-p53 interaction, and we elucidated preliminary structure–activity relationships. One of the identified compounds had the potential to inhibit the S100B–p53 interaction in melanoma cells.     

The N-terminal domain of elastin-binding protein of Staphylococcus aureus changes its secondary structure in a membrane-mimetic environment

Elastin-binding protein of Staphylococcus aureus (EbpS) has been identified as an adhesin that can bind to soluble elastin or tropoelastin. However, the structure and exact function of EbpS remain to be elucidated. To gain insight into the molecular characteristics of EbpS, we investigated the physical properties of its N-terminal extracellular domain in various environments. CD spectroscopy showed that this protein was soluble and unstructured under aqueous conditions. Non-native secondary structures, however, were induced by several alcohols that provided membrane-mimetic environments. These changes may have some correlation with the function of this protein.     

Mimicry of erythropoietin and interleukin-6 signalling by an antibody/cytokine receptor chimera in murine myeloid 32D cells

We have previously designed antibody-cytokine receptor chimeras that could respond to a cognate antigen. While these chimeric receptors were functional, it has not been investigated exactly how they mimic signal transduction through corresponding wild-type receptors. In this study, we compared the growth properties and the phosphorylation status of intracellular signal transducers between the erythropoietin receptor (EpoR)- or gp130-based chimeric receptors and wild-type EpoR or EpoR-gp130 chimera, respectively. Expression plasmids, encoding V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2 domain of EpoR and transmembrane/cytoplasmic domains of either EpoR or gp130, were constructed, and pairs of chimeric receptor combinations (V(H)-EpoR and V(L)-EpoR, V(H)-gp130 and V(L)-gp130, V(H)-EpoR and V(L)-gp130, V(H)-gp130 and V(L)-EpoR) were expressed in an IL-3-dependent myeloid cell line, 32D. Growth assay revealed that the transfectants all grew in a HEL-dependent manner. As for phosphorylation of Stat3, Stat5, ERK and Akt, the chimeric receptors showed similar activation pattern of signalling molecules with wild-type receptors, although the chimeric receptors showed ligand-independency and a little lower maximal phosphorylation than the corresponding wild-type receptors. The results demonstrate that antibody-receptor chimeras could substantially mimic wild-type receptors.     

Suppression of protein interactions by arginine: a proposed mechanism of the arginine effects

Arginine has been used to suppress protein aggregation and protein-protein or protein-surface interactions during protein refolding and purification. While its biotechnology applications are gradually expanding, the mechanism of these effects of arginine has not been fully elucidated. Arginine is more effective at higher concentrations, an indication of weak interactions with the proteins. The effects of weakly interacting additives, such as arginine, on protein solubility, stability and aggregation have been explained from three different approaches: i.e., (1) the effects of additives on the structure of water, (2) the interactions of additives with the amino acid side chains and peptide bonds and (3) the preferential interactions of additives with the proteins. Here we have examined these properties of arginine and compared with those of other additives, e.g., guanidine hydrochloride (GdnHCl) and certain amino acids and amines. GdnHCl is a strong salting-in agent and denatures proteins, while betaine is a protein stabilizer. Several amino acids and amine compounds, including betaine, which stabilize the proteins, are strongly excluded; i.e., the proteins are preferentially hydrated in these solutions. On the other hand, GdnHCl preferentially binds to the proteins. Arginine is intermediate between these two extreme cases and shows a more complicated pattern of interactions with the proteins. The effects of additives on water structure, e.g., the surface tension of aqueous solution of the additives and the solubility of amino acids in the presence of additives also shed light on the mechanism of the effects of the additives on protein aggregation. While arginine increases the surface tension of water, it favorably interacts with most amino acid side chains and the peptide bonds, a property shared with GdnHCl. Thus, we propose that while arginine is similar to GdnHCl in the amino acid level, arginine interacts with the proteins differently from GdnHCl.     

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.