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Nearly fifty monoclonal antibodies have been approved to date, and the market for monoclonal antibodies is expected to continue to grow. Since global competition in the field of antibody therapeutics is intense, we need to establish novel antibody engineering technologies to provide true benefit for patients, with differentiated product values. Bispecific antibodies are among the next generation of antibody therapeutics that can bind to two different target antigens by the two arms of immunoglobulin G (IgG) molecule, and are thus believed to be applicable to various therapeutic needs. Until recently, large scale manufacturing of human IgG bispecific antibody was impossible. We have established a technology, named asymmetric re-engineering technology (ART)-Ig, to enable large scale manufacturing of bispecific antibodies. Three examples of next generation antibody therapeutics using ART-Ig technology are described. Recent updates on bispecific antibodies against factor IXa and factor X for the treatment of hemophilia A, bispecific antibodies against a tumor specific antigen and T cell surface marker CD3 for cancer immunotherapy, and bispecific antibodies against two different epitopes of soluble antigen with pH-dependent binding property for the elimination of soluble antigen from plasma are also described.

G protein-coupled receptors (GPCRs) are major therapeutic drug targets and represent more than 30% of the market share of all prescription drugs. The high-resolution three-dimensional structures of the target receptors provide good initial models for structure-based approaches to drug screening and drug design, which are considered to accelerate drug discovery. However, significant bottlenecks at the expression, purification and crystallization stages of structure determination of GPCRs have existed. Here, we review recent techniques for the determination of GPCR structures. In particular, we focus on the protein engineering techniques that have been used to overcome bottlenecks in expression/purification and crystallization, including our development of a platform using budding yeast for the rapid construction and evaluation of GPCR variants for structural studies. We also present our success in determining the crystal structure of the histamine H₁ receptor (H₁R) in complex with doxepin, an inverse agonist antihistamine. The H₁R structure revealed the low selectivity of doxepin to aminergic receptors and provides key information that should aid the development of highly selective antihistamines.

We have seen increasing use of the term "White biotechnology". White biotechnology involves the use of microbial cells and enzymes in the production of bulk and fine chemicals such as amino acids and polymers. This generally results in cleaner processes with minimum waste generation and energy use. Most of the organic syntheses using enzymes are carried out in nearly anhydrous organic solvents or solvent-free media. Ionic liquids have more recently emerged as another nonaqueous media, which, in view of their low vapor pressure, are viewed as "green solvents". Organic solvents may alter the structure and activity of enzymes that usually function in an aqueous environment. One alternative is to immobilize the enzymes on solid supports to increase their function and stability in response to organic solvents or increased temperatures. Enzymes may be stabilized by chemical and physical processes. With chemical methods, enzymes are immobilized by strong covalent bonding, but changes in protein structure often result. In physical stabilization processes, the interactions between enzymes and solids usually are weaker, resulting in fewer changes in the enzyme's structure. Yeast cell surface engineering is an alternative approach that immobilizes enzymes on the yeast cell surface. Proteins are immobilized by using an outer shell cell-wall protein, the C-terminal half of alpha-agglutinin. Display of enzymes on the yeast cell surface has at least two advantages relative to other physical immobilization methods. First, the displayed enzymes can be readily produced in a standard fermentation. No further work is required to either purify or immobilize the enzymes. Second, enzyme displayed on the yeast cell surface can be modified directly by conventional genetic engineering, which enables error-prone PCR, DNA shuffling, and combinatorial mutagenesis to be used quickly and efficiently to create strains (whole-cell biocatalysts) with enhanced enzyme activity.

Techniques for immobilizing proteins on surface of virus or microorganisms, namely molecular display technologies, have played important roles in helping the elucidation of protein-protein interactions in cells and to develop research on drug discovery. Phage display system is well-established and sophisticated; consequently, bioactive low-molecular-weight ligands and proteins significant in pharmaceutical industry have been found. In addition to the development of novel functional proteins by phage display using results from experiments in genomics and proteomics, ribosome display or yeast display systems have been developed as complementary methods. We can select the appropriate method on the basis of the objective. Molecular display using yeast has advantages in production of desired proteins from combinatorial library by flow cytometry. Firstly, principle, development procedure, and latest research in this field are introduced. Thereafter, results of molecular display using yeast for antibodies and their related proteins are presented. Furthermore, display of receptor coupled with intracellular signal transduction -a novel type of molecular display on yeast cell surface- has been created in recent years. The role and potential of molecular display technologies employing yeast cells in drug discovery are discussed.

Magnetic particles offer vast potential in ushering new techniques, especially in biomedical applications, as they can be easily manipulated by magnetic force. Magnetotactic bacteria synthesize nano-sized biomagnetites, otherwise known as bacterial magnetic particles (BacMPs) that are individually enveloped by a lipid bilayer membrane. BacMPs are ultrafine magnetite crystals (50-100 nm diameters) with uniform morphology produced by Magnetospirillum magneticum AMB-1. Based on our elucidations on the molecular mechanism of BacMP formation in M. magneticum AMB-1, functional nanomaterials have been designed. Through genetic engineering, functional proteins such as enzymes, antibodies, and receptors were successfully displayed onto BacMPs. Here, display techniques of functional proteins onto nano-sized BacMPs and its applications to ligand binding assays were described. Dopamine receptor, which is a member of G protein-coupled receptors, was successfully displayed onto BacMPs. This system makes possible the convenient acquisition of the native conformation of membrane proteins without the need for detergent solubilization, purification and reconstitution after cell disruption. Furthermore, estrogen receptor, which is one of nuclear receptors, was also displayed onto BacMPs. The assay using BacMPs displaying estrogen receptor could discriminate full agonists, partial agonists, or antagonists. The elucidation of the mechanism of BacMP synthesis has provided a roadmap for the design of novel nano-biomaterials that would play a useful role in multidisciplinary fields.

Monoclonal antibodies (MAbs) have been utilized as research tools, as diagnostic reagents, and for antibody medicine. The preparation of MAbs involves laborious processes and normally takes months. Here we describe an ex vivo B cell-based antibody display system called the ADLib (Autonomously Diversifying Library) system, which enables us to select chicken B cell clones producing antibody against antigens of interest in a couple of weeks. The ADLib system is applicable to self- or highly conserved antigens, polysaccharide chains, peptides, and haptens.

Advances in methods for conformational prediction, structural analysis and site-directed mutagenesis of proteins and peptides have contributed to the understanding of their structure and function. However, with the exception of a few successes, the generation of practical functional molecules solely by rational design remains a difficult challenge. The aim of our study is to investigate molecular design relying on evolutionary processes, called as "directed evolution", to generate a novel class of biofunctional molecules. This evolutionary approach consists of three steps; 1) constructions of protein/peptide libraries based on structural information, 2) expressions of the libraries on phage particles, and 3) selections with investigator-imposed selective pressures. In this work, we study on directed evolution with antibody libraries. We have succeeded in generating highly active catalytic antibodies in phage-displayed antibody (Fab) libraries. To evolve catalytic antibodies toward higher catalytic activity, we have mimicked an enzyme-evolutional process, in which an enzyme has evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. Thus, phage-displayed libraries derived from an original catalytic antibody were selected against a newly-devised TSA, which was programmed to optimize the differential affinity for the transition state relative to the ground state, to provide variants with improved reaction rates (k(cat)). The in vitro evolution has great potential for generating novel catalysts as well as for providing opportunities to examine the evolutionary dynamics of enzymes.

The recent development of molecular biology has been elucidating outlines of the cross-talk of biomolecules. The understanding of the function of these biomolecules from the viewpoint of chemistry is now demanded not only for the understanding of biological systems but also for the creation of novel functional molecules. Here two topics are described about peptide synthesis aiming at the elucidation and the creation of protein functions. The first topic is the development of approaches for the synthesis of Tyr (SO3H)-containing peptides. Tyrosine sulfation is one of the most popular protein post-translational modifications. Synthetic peptides are of great help for the elucidation of the biological significance of tyrosine sulfation. We have developed two approaches for the efficient synthesis of tyrosine sulfate [Tyr (SO3H)]-containing peptides. The first approach employs a dimethylformamide-sulfur trioxide (DMF-SO3) complex as a sulfating agent and safety-catch protecting groups for the selective sulfation of tyrosine in the presence of serine. The second approach employs the direct introduction of Tyr(SO3H) into the peptide chain in the form of Fmoc-Tyr(SO3Na) followed by deprotection at 4 degrees C in trifluoroacetic acid. These approaches were successfully applied for the synthesis of cholecystokinin (CCK)-related peptides. The second topic deals with new approaches for the creation of artificial proteins through assembling alpha-helical peptides via selective disulfide or thioether formation. Approaches to assemble individual peptide segments on a peptide template were also developed. Four peptides corresponding to the transmembrane segments of the sodium channel (S4 in repeat I-IV) were assembled on a peptide template to give a protein having ion channel activity with rectification.